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**__MY NEW NOTEBOOK (as of 10/19/2011):__**
===[|https://www.evernote.com/pub/sammibrombacker/worknotebook#b=d2d758aa-8546-49f5-beb0-991a6f900d58&n=0590b692-de86-4bfb-a603-a5fa2c0b90cf]===

__**9/29/2011: Laby MBL 93 Gel**__

GEL PREP:


 * 1) Weigh 2g of agarose and mix with 150mL 1x TBE in a 500 mL flask
 * 2) Microwave solution for ~ 3 minutes
 * 3) Cool solution (you should be able to touch the flask for a few seconds), then add 12uL ethidium bromide(EtBr). WARNING: EtBr is a carcinogen be sure to wear gloves and appropriately dispose tip waste.
 * 4) Mix thoroughly by swirling, then pour into gel tray.
 * 5) Add gel combs

ELECTROPHORESIS PROCEDURE:
 * 1) Place gel in gel box and fill with 1x TBE buffer (to fully cover wells)
 * 2) Remove combs from wells
 * 3) Load 1uL 100bp ladder + 1 uL loading dye + 4 uL H2O in far left lane on both top and bottom
 * 4) Load 5uL of your PCR sample + 1 uL of loading dye into the gel
 * 5) Run gel at ~ 110V for ~ 2hr
 * 6) Visualize the gel on the UV transilluminator



__**9/28/2011**__

Muk for:
 * seawater
 * larval care
 * clean lab tanks
 * minor fixes

Changed H2O and filters in basement

Cleaned filters

Helped Liza set up her experiment

__**9/27/2011: Laby MBL 93 PCR and WSN qPCR**__

LABY PCR:

Ran the following PCR for each MBL primer I have (n=3) with the new annealing temp of 50 deg C

__PCR__ Recipe per rxn (uL):
 * 12.5 Immo
 * 1.5 BSA
 * 0.8 Fwd primer
 * 0.8 Reverse primer
 * 7.4 H2O
 * 2.0 template

Master Mixes (10 rxns...uL):
 * 125 Immo
 * 15 BSA
 * 8 Fwd primer
 * 8 Reverse primer
 * 74 H2O

Profile:
 * Time |||| Temp (°C) ||
 * Step 1 |||| 10 min || 95 ||
 * Step 2 |||| 30 min || 95 ||
 * Step 3 |||| 30 sec || 50 ||
 * Step 4 |||| 30 sec || 72 ||
 * Repeat steps 2-4, 40 times ||
 * Step 5 |||| 10 min || 72 ||

Stored PCR product in fridge till I can run gel



WS qPCR

I ran the fecal samples taken from the Big Reds along with the filters that Anne extracted at NOAA

//Results:// The ran went really well other than the plasmids are possibly getting old. Blacks are clean :) As for infection level: Round Reds < Red 1 < Big Reds



__**9/26/2011: Seawater from Muk & procard reconciliation**__

__**8/9/2011: Laby nested PCR results**__

ELECTROPHORESIS PROCEDURE: > Results:
 * 1) <span style="font-size: 12px; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Place gel in gel box and fill with 1x TBE buffer (to fully cover wells)
 * 2) <span style="font-size: 12px; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Remove combs from wells
 * 3) <span style="font-size: 12px; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Load 1uL 100bp ladder + 1 uL loading dye + 4 uL H2O in far left lane on both top and bottom
 * 4) <span style="font-size: 12px; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Load 5uL of your PCR sample + 1 uL of loading dye into the gel
 * 5) <span style="font-size: 12px; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Run gel at ~ 110V for ~ 2hr
 * 6) <span style="font-size: 12px; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Visualize the gel on the UV transilluminator

__**8/8/2011: Muk checks, cleaned 236, Laby gel prep**__

Gel Prep:
 * 1) Weigh 2g of agarose and mix with 150mL 1x TBE in a 500 mL flask
 * 2) Microwave solution for ~ 3 minutes
 * 3) Cool solution (you should be able to touch the flask for a few seconds), then add 12uL ethidium bromide(EtBr). WARNING: EtBr is a carcinogen be sure to wear gloves and appropriately dispose tip waste.
 * 4) Mix thoroughly by swirling, then pour into gel tray.
 * 5) Add gel combs
 * 6) Wrap in foil ant store at 4 until tomorrow morning

__**7/27/2011: Muk checks, PCR - Laby, artificial feed**__


 * Muk checks:** checked in on Muk, fed more art feed to treatment group, set up for spawn, made spawning chemicals


 * Basement checks:** daily checks, fed animals, & moved shakers

Using the following PCR protocol, I re-PCRd my product from yesterday for all MBL 93-2 samples in hopes to make the faint electrophoresis results stronger
 * Laby PCR:**

__PCR__ Recipe per rxn (uL):
 * <span style="font-family: Arial,sans-serif;">12.5 Immo
 * <span style="font-family: Arial,sans-serif;">1.5 BSA
 * <span style="font-family: Arial,sans-serif;">0.8 Fwd primer
 * <span style="font-family: Arial,sans-serif;">0.8 Reverse primer
 * <span style="font-family: Arial,sans-serif;">7.4 H2O
 * <span style="font-family: Arial,sans-serif;">2.0 template [PCR product from 7/26 PCR reaction]

<span style="font-family: Arial,sans-serif;">Master Mixes (10 rxns...uL):
 * <span style="font-family: Arial,sans-serif;">125 Immo
 * <span style="font-family: Arial,sans-serif;">15 BSA
 * <span style="font-family: Arial,sans-serif;">8 Fwd primer
 * <span style="font-family: Arial,sans-serif;">8 Reverse primer
 * <span style="font-family: Arial,sans-serif;">74 H2O

Profile:
 * Time |||| Temp (°C) ||
 * Step 1 |||| 10 min || 95 ||
 * Step 2 |||| 30 min || 95 ||
 * Step 3 |||| 30 sec || 53 deg ||
 * Step 4 |||| 30 sec || 72 ||
 * Repeat steps 2-4, 40 times ||
 * Step 5 |||| 10 min || 72 ||

Mixed dry ingredients above and mixed into to 2400 mL. Poured into tray and placed in -80 freezer @ 4:00 pm.
 * Artificial feed:**
 * Ingredient || Amount (g) ||
 * yellow corn flour || 26 ||
 * kelp flour || 26 ||
 * soy flour || 26 ||
 * whole wheat flour || 26 ||
 * Spirulina || 8 ||
 * 1.3 mm screened fish meal || 96 ||
 * Manugel DMB || 36 ||

__**7/26/2011: Replate Laby cultures, PCR - Laby, and electrophoresis**__

All cultures replated from original plates Sandy brought down on 5/26 which have been stored at 4 deg

__Aliquot and Dilute Primers__ 10 uL primer + 90 uL Low TE

__PCR__ Recipe per rxn (uL):
 * <span style="font-family: Arial,sans-serif;">12.5 Immo
 * <span style="font-family: Arial,sans-serif;">1.5 BSA
 * <span style="font-family: Arial,sans-serif;">0.8 Fwd primer
 * <span style="font-family: Arial,sans-serif;">0.8 Reverse primer
 * <span style="font-family: Arial,sans-serif;">7.4 H2O
 * <span style="font-family: Arial,sans-serif;">2.0 template

<span style="font-family: Arial,sans-serif;">Master Mixes (10 rxns...uL):
 * <span style="font-family: Arial,sans-serif;">125 Immo
 * <span style="font-family: Arial,sans-serif;">15 BSA
 * <span style="font-family: Arial,sans-serif;">8 Fwd primer
 * <span style="font-family: Arial,sans-serif;">8 Reverse primer
 * <span style="font-family: Arial,sans-serif;">74 H2O

Profile:
 * Time |||| Temp (°C) ||
 * Step 1 |||| 10 min || 95 ||
 * Step 2 |||| 30 min || 95 ||
 * Step 3 |||| 30 sec || ** 154- 60 deg MBL 93-2 - 53 deg ** ||
 * Step 4 |||| 30 sec || 72 ||
 * Repeat steps 2-4, 40 times ||
 * Step 5 |||| 10 min || 72 ||

Gel Prep:
 * 1) Weigh 2g of agarose and mix with 150mL 1x TBE in a 500 mL flask
 * 2) Microwave solution for ~ 3 minutes
 * 3) Cool solution (you should be able to touch the flask for a few seconds), then add 12uL ethidium bromide(EtBr). WARNING: EtBr is a carcinogen be sure to wear gloves and appropriately dispose tip waste.
 * 4) Mix thoroughly by swirling, then pour into gel tray.
 * 5) Add gel combs

ELECTROPHORESIS PROCEDURE:
 * 1) Place gel in gel box and fill with 1x TBE buffer (to fully cover wells)
 * 2) Remove combs from wells
 * 3) Load 1uL 100bp ladder + 1 uL loading dye + 4 uL H2O in far left lane on both top and bottom
 * 4) Load 5uL of your PCR sample + 1 uL of loading dye into the gel
 * 5) Run gel at ~ 110V for ~ 2hr
 * 6) Visualize the gel on the UV transilluminator

Results:



__**7/25/2011: Plates for Laby exp**__

**<span style="color: black; font-family: Arial,sans-serif; font-size: 10.5pt;">SSA PLATES ** <span style="font-family: Arial,sans-serif; font-size: 14px; line-height: 21px;">1 Liter 0.45um filtered seawater, adjusted to 25ppt <span class="apple-style-span" style="color: black; font-family: Arial,sans-serif; font-size: 10.5pt;">"USB Nobel <span style="color: black; font-family: Arial,sans-serif; font-size: 10.5pt;">": 12g <span class="apple-style-span" style="color: black; font-family: Arial,sans-serif; font-size: 10.5pt;">Horse Serum: 10mL <span class="apple-style-span" style="color: black; font-family: Arial,sans-serif; font-size: 10.5pt;">Pen/Strep: 25mL <span class="apple-style-span" style="color: black; font-family: Arial,sans-serif; font-size: 10.5pt;">Germanium dioxide: 1.5mg

<span style="color: black; font-family: Arial,sans-serif; font-size: 10pt;">10 g NaCl <span style="color: black; font-family: Arial,sans-serif; font-size: 10pt;"> 10 g Bactotryptone <span style="color: black; font-family: Arial,sans-serif; font-size: 10pt;"> 5 g Yeast Extract <span style="color: black; font-family: Arial,sans-serif; font-size: 10pt;"> Bring to 800 ml with water and stir on stir plate <span style="color: black; font-family: Arial,sans-serif; font-size: 10pt;"> Adjust pH to 7.0 <span style="color: black; font-family: Arial,sans-serif; font-size: 10pt;"> Add 20 g Bacto Agar
 * LB Plates**

__**6/24/2011: Pinto feeding trial, new system set-up, RNA later for Kathy**__

//Filled 79 cryovials with 500 uL RNA later and then shipped to Kathy//

//Helped Lisa set up second tank for new big reds//

//Worked on BS ab feed using freeze drying method://
 * Ingredient || Amount (g) ||
 * yellow corn flour || 24 ||
 * kelp flour || 26 ||
 * soy flour ||  ||
 * whole wheat flour || 26 ||
 * Spirulina || 8 ||
 * 1.3 mm screened fish meal || 96 ||
 * Manugel DMB || 36 ||

Mixed dry ingredients above and mixed into to 2400 mL. Poured into tray and placed in -80 freezer @ 1:30 pm.





__**6/24/2011**__

//Seawater run//

//H20 changes in basement//

//Processed pinto ab DNA with new primers using ABI macine @ Mukilteo with Jon//



Jon will email final results early next week.

//Muk checks//

__**6/22/2011**__

//Ran PCR on practice pinto tissue using new primers [redo from 6/20/2011]//


 * __Master Mix:__**
 * <span style="color: black; font-family: Arial,sans-serif; font-size: 10pt;">Sensimix Probe II - 50 uL
 * <span style="color: black; font-family: Arial,sans-serif; font-size: 10pt;">PCR Water - 25 uL
 * <span style="color: black; font-family: Arial,sans-serif; font-size: 10pt;">F primer - 5 uL
 * <span style="color: black; font-family: Arial,sans-serif; font-size: 10pt;">R primer - 5 uL
 * <span style="color: black; font-family: Arial,sans-serif; font-size: 10pt;">BSA - 5 uL

<span style="color: black; font-family: Arial,sans-serif; font-size: 10pt;">9.0 uL MM + 1.0 uL = 10.0 uL per well
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">Plate 1 on Cycle HKA1 ** ||
 * || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;"> 1 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">2 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">3 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">4 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">5 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">6 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">7 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">8 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">9 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">10 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">11 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">12 ** ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">E ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA12 08:08-1 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA12 08:08-2 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA12 08:08-3 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA12 08:08-4 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA12 08:08-5 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA12 08:08-6 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA12 08:08-7 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA12 08:08-8 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA40 08:08-1 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA40 08:08-2 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA40 08:08-3 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA40 08:08-4 ** ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">F ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA40 08:08-5 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA40 08:08-6 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA40 08:08-7 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA40 08:08-8 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA48 08:08-1 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA48 08:08-2 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA48 08:08-3 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA48 08:08-4 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA48 08:08-5 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA48 08:08-6 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA48 08:08-7 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA48 08:08-8 ** ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">G ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA65 08:08-1 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA65 08:08-2 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA65 08:08-3 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA65 08:08-4 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA65 08:08-5 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA65 08:08-6 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA65 08:08-7 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA65 08:08-8 ** ||  ||   ||   ||   ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">H ** ||  ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||


 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">Plate 2 on Cycle HKA2 ** ||  ||
 * || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;"> 5 ** ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">A ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA43 08:08-1 ** ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">B ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA43 08:08-2 ** ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">C ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA43 08:08-3 ** ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">D ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA43 08:08-4 ** ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">E ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA43 08:08-5 ** ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">F ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA43 08:08-6 ** ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">G ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA43 08:08-7 ** ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">H ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA43 08:08-8 ** ||


 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">Plate 3 on Cycle HKA3 ** ||
 * || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">7 ** ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">A ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA56 08:08-1 ** ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">B ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA56 08:08-2 ** ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">C ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA56 08:08-3 ** ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">D ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA56 08:08-4 ** ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">E ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA56 08:08-5 ** ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">F ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA56 08:08-6 ** ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">G ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA56 08:08-7 ** ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">H ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA56 08:08-8 ** ||

__**6/21/2011: Muk day**__

__**6/20/2011**__

//Ran PCR on practice pinto tissue using new primers...will have to redo pcr's...didn't aliquot primers correctly//

<span style="color: black; font-family: Arial,sans-serif; font-size: 10pt;">9.0 uL MM + 1.0 uL = 10.0 uL per well
 * __Master Mix:__**
 * <span style="color: black; font-family: Arial,sans-serif; font-size: 10pt;">Sensimix Probe II - 50 uL
 * <span style="color: black; font-family: Arial,sans-serif; font-size: 10pt;">PCR Water - 25 uL
 * <span style="color: black; font-family: Arial,sans-serif; font-size: 10pt;">F primer - 5 uL
 * <span style="color: black; font-family: Arial,sans-serif; font-size: 10pt;">R primer - 5 uL
 * <span style="color: black; font-family: Arial,sans-serif; font-size: 10pt;">BSA - 5 uL
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">Plate 1 on Cycle HKA1 ** ||
 * || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;"> 1 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">2 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">3 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">4 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">5 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">6 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">7 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">8 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">9 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">10 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">11 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">12 ** ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">A ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA12 08:08-1 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA12 08:08-2 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA12 08:08-3 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA12 08:08-4 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA12 08:08-5 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA12 08:08-6 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA12 08:08-7 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA12 08:08-8 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA40 08:08-1 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA40 08:08-2 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA40 08:08-3 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA40 08:08-4 ** ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">B ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA40 08:08-5 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA40 08:08-6 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA40 08:08-7 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA40 08:08-8 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA48 08:08-1 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA48 08:08-2 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA48 08:08-3 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA48 08:08-4 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA48 08:08-5 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA48 08:08-6 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA48 08:08-7 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA48 08:08-8 ** ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">C ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA65 08:08-1 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA65 08:08-2 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA65 08:08-3 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA65 08:08-4 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA65 08:08-5 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA65 08:08-6 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA65 08:08-7 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA65 08:08-8 ** ||  ||   ||   ||   ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">D ** ||  ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||


 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">Plate 2 on Cycle HKA2 ** ||
 * || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;"> 1 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">2 ** ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">A ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA43 08:08-1 ** ||  ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">B ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA43 08:08-2 ** ||  ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">C ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA43 08:08-3 ** ||  ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">D ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA43 08:08-4 ** ||  ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">E ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA43 08:08-5 ** ||  ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">F ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA43 08:08-6 ** ||  ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">G ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA43 08:08-7 ** ||  ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">H ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA43 08:08-8 ** ||  ||


 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">Plate 3 on Cycle HKA3 ** ||
 * || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">3 ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">4 ** ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;"> A ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA56 08:08-1 ** ||  ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">B ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA56 08:08-2 ** ||  ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">C ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA56 08:08-3 ** ||  ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">D ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA56 08:08-4 ** ||  ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">E ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA56 08:08-5 ** ||  ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">F ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA56 08:08-6 ** ||  ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">G ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA56 08:08-7 ** ||  ||
 * **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">H ** || **<span style="font-family: 'Times New Roman',serif; font-size: 10pt;">HKA56 08:08-8 ** ||  ||

//Ordered Laby primers for MBL 93-23//

__**6/16/2011: Laby primers**__

Ran a ClusterM alignment to decipher between E. coast and. coast strain:

code
 * 1) Program: needle
 * 2) Rundate: Fri 17 Jun 2011 18:03:26
 * 3) Commandline: needle
 * 4)    -auto
 * 5)    -stdout
 * 6)    -asequence emboss_needle-E20110617-180325-0731-59458571-pg.asequence
 * 7)    -bsequence emboss_needle-E20110617-180325-0731-59458571-pg.bsequence
 * 8)    -datafile EDNAFULL
 * 9)    -gapopen 10.0
 * 10)    -gapextend 0.5
 * 11)    -endopen 10.0
 * 12)    -endextend 0.5
 * 13)    -aformat3 pair
 * 14)    -snucleotide1
 * 15)    -snucleotide2
 * 16) Align_format: pair
 * 17) Report_file: stdout
 * 1) Report_file: stdout


 * 1) Aligned_sequences: 2
 * 1: EMBOSS_001 - TYPE_154
 * 2: EMBOSS_001 - MBL 93-2
 * 1) Matrix: EDNAFULL
 * 2) Gap_penalty: 10.0
 * 3) Extend_penalty: 0.5
 * 4) Length: 1179
 * 5) Identity:    1129/1179 (95.8%)
 * 6) Similarity:  1129/1179 (95.8%)
 * 7) Gaps:          16/1179 ( 1.4%)
 * 8) Score: 5435.0
 * 1) Similarity:  1129/1179 (95.8%)
 * 2) Gaps:          16/1179 ( 1.4%)
 * 3) Score: 5435.0

EMBOSS_001        1 -anccntggttgattctgccagtagtcatacgcttgttcaaaagattaag     49 | || |||||||||||||||||||.|||||||||||||||||||||||| EMBOSS_001        1 ta-ccctggttgattctgccagtagacatacgcttgttcaaaagattaag     49

EMBOSS_001       50 ccatgcaagtgtaagctcatatgttctattttgaagctgcgaatggctca     99 |||||||||||||||||||||||||||||||||||||||||||||||||| EMBOSS_001       50 ccatgcaagtgtaagctcatatgttctattttgaagctgcgaatggctca     99

EMBOSS_001      100 ttatatcagtcacaacttaatctacgacacaatatggataactgtagtaa    149 |||||||||||||||||||||||||||||||||||||||||||||||||| EMBOSS_001      100 ttatatcagtcacaacttaatctacgacacaatatggataactgtagtaa    149

EMBOSS_001      150 ttctagagctaatacatggaacacctatcggtacatttattagattgaaa    199 |||||||||||||||||||||||||||||||||||||||||||||||||| EMBOSS_001      150 ttctagagctaatacatggaacacctatcggtacatttattagattgaaa    199

EMBOSS_001      200 ctgggaaaacgttgattcaaaataaagctgtaaatcgcatgcgatatgta    249 |||||||||||||||||||||||||||||||||||||||||||||||||| EMBOSS_001      200 ctgggaaaacgttgattcaaaataaagctgtaaatcgcatgcgatatgta    249

EMBOSS_001      250 gactaggcttctgacctatcagctgtcgatggtaaggtattggcttacca    299 |||||||||||||||||||||||||||||||||||||||||||||||||| EMBOSS_001      250 gactaggcttctgacctatcagctgtcgatggtaaggtattggcttacca    299

EMBOSS_001      300 tggcgttaacgggtaacggggaattagggttcgattccggagagggagcc    349 |||||||||||||||||||||||||||||||||||||||||||||||||| EMBOSS_001      300 tggcgttaacgggtaacggggaattagggttcgattccggagagggagcc    349

EMBOSS_001      350 tgagagacggctaccacatccaaggaaggcagcaggcgcgtaaattaccc    399 |||||||||||||||||||||||||||||||||||||||||||||||||| EMBOSS_001      350 tgagagacggctaccacatccaaggaaggcagcaggcgcgtaaattaccc    399

EMBOSS_001      400 aatcctgatacggggaggtagtgacagaaaatagcaatacaagacctatt    449 |||||||||||||||||||||||||||||||||||||||||||||||||| EMBOSS_001      400 aatcctgatacggggaggtagtgacagaaaatagcaatacaagacctatt    449

EMBOSS_001      450 ggttttgtaattggaatgagttaaatataaaatttttgattaggaacaat    499 |||||||||||||||||||||||||||||||||||||||||||||||||| EMBOSS_001      450 ggttttgtaattggaatgagttaaatataaaatttttgattaggaacaat    499

EMBOSS_001      500 tgga--ggaccgnaaatagcaaaaccagaccta-tggttttgtaattgga    546 |||| ||   . |||||||||.||.||||||| |||||||||||||||| EMBOSS_001       500 tggantgg---ngaaatagcaatacaagacctattggttttgtaattgga    546

EMBOSS_001      547 atgagttaaatataaaattttggattaggaaccaattggg__//**agccaagtct**//__    596 |||||||||||||||||||||.||||||||| |||||||..|.||||||| EMBOSS_001      547 atgagttaaatataaaatttttgattaggaa-caattggagggcaagtct    595

EMBOSS_001      597 __//**ggtgccagcag**//__ccgcgggaaaaccagctccagtagcgtatattaaagttg    646 |||||||||||||.|||||||.|||||||||||||||||||||||||||| EMBOSS_001      596 ggtgccagcagccacgggaaatccagctccagtagcgtatattaaagttg    645

EMBOSS_001      647 ttgcagttaaaaagctcgtagttgaaagtctggagatgatttcttttgat    696 |||||||||||||||||||||||||||||||||||||||||||||||||| EMBOSS_001      646 ttgcagttaaaaagctcgtagttgaaagtctggagatgatttcttttgat    695

EMBOSS_001      697 acttcaattgatttctgacccattttgcaggagtcttcttcgcattaact    746 |||||||||||||||||||||||||||||||.|||||||||||||||||| EMBOSS_001      696 acttcaattgatttctgacccattttgcaggggtcttcttcgcattaact    745

EMBOSS_001      747 tgtggggtcgtacgaatgtagcgtttactgtgagaaaattagggtgttta    796 ||||||||||||||||||||||||.||||||||||||||||||||||||| EMBOSS_001      746 tgtggggtcgtacgaatgtagcgtatactgtgagaaaattagggtgttta    795

EMBOSS_001      797 aagcaagcgagtgtgtgaatacattagcatggaataataagacatagttc    846 |||||||||||||||||||||||||||||||||||||||||||||||||| EMBOSS_001      796 aagcaagcgagtgtgtgaatacattagcatggaataataagacatagttc    845

EMBOSS_001      847 ggcatactgtct-ctcagtttgcaatgctggataatgattaaaaggaact    895 |||||||||||| ||||||||||||||||||||||||||||||||||||| EMBOSS_001      846 ggcatactgtctactcagtttgcaatgctggataatgattaaaaggaact    895

EMBOSS_001      896 gatgtgggtattcgtatgagtatgtcagaggtgaaattcttggattttat    945 |||||||||||||||||||||||||||||||||||||||||||||||||| EMBOSS_001      896 gatgtgggtattcgtatgagtatgtcagaggtgaaattcttggattttat    945

EMBOSS_001      946 tcagacggtctacagcgaaggcatttaccaaagatgttttcattaatcaa    995 |||||||||||||||||||||||||||||||||||||||||||||||||| EMBOSS_001      946 tcagacggtctacagcgaaggcatttaccaaagatgttttcattaatcaa    995

EMBOSS_001      996 gaacgaaagttaggggatcgaagatgcttagataccatcgtagtcttaac   1045 |||||||||||||||||||||||||||||||||||||||||||||||||| EMBOSS_001      996 gaacgaaagttaggggatcgaagatgcttagataccatcgtagtcttaac   1045

EMBOSS_001     1046 cataaactataccgactgaggattggaaacattcataatgggtgttttca   1095 |||||||||||||||||||||||||||||||||||.||.|||.||||..| EMBOSS_001     1046 cataaactataccgactgaggattggaaacattcaaaaggggggtttcaa   1095

EMBOSS_001     1096 aaactcnttcgagaaatcaaagtttttgggttccgg//__**ggggaatatggtca**__//   1145                     |||  | |||.|.|||.||||..|||||||.|||.|||||||.|.||.|| EMBOSS_001     1096 aaa--ccttccaaaaaacaaaagttttggggtcccgggggaagaagggca   1143

EMBOSS_001     1146 //__**caaggctgaaaa**__//tgaaaggaattgac---   1171 .||.||.| ||||.|||||||||||| EMBOSS_001     1144 aaaagccg-aaattaaaggaattgacggg   1171

code

Conclusion: Too similar to find specific primers for each. Decied to just follow through with the W. coast strain (typer-154)

Ordering the following primers for Type-154 (West coast) [GenBank: AF265335.1 [] ]

Primer pair 9
Products on intended target
 * ~  ||~ Sequence (5'->3') ||~ Strand on template ||~ Length ||~ Start ||~ Stop ||~ Tm ||~ GC% ||
 * ~ Forward primer || AGCCAAGTCTGGTGCCAGCAG || Plus || 21 || 587 || 607 || 59.31 || 61.90% ||
 * ~ Reverse primer || TTTTCAGCCTTGTGACCATATTCCCC || Minus || 26 || 1157 || 1132 || 57.36 || 46.15% ||
 * ~ Internal oligo ||  || Plus ||   ||   ||   ||   ||   ||
 * ~ Product length |||||||||||| 571 ||
 * ~ Product Tm ||||||||||||  ||
 * ~ Product Tm - min(OLIGO Tm) ||||||||||||  ||
 * ~ Exon junction ||||||||||||||||  ||
 * ~ Total intron size ||||||||||||||||  ||

> AF265335.1 Labyrinthula zosterae type-154 small subunit ribosomal RNA gene, partial sequence code product length = 571 Forward primer 1 AGCCAAGTCTGGTGCCAGCAG 21 Template 587 ..................... 607

Reverse primer 1 TTTTCAGCCTTGTGACCATATTCCCC 26 Template 1157 .......................... 1132

__**6/15/2011: Pinto BS Extractions**__

Extracted Mukilteo BS Y15 through Y37 using the Qiagen DNeasy Blood and Stool Kit. Followed manufacturer's animal tissue extraction protocol with the addition of the additional 200 uL AE rinse. The incubation step lasted for 3 hrs.

__**6/14/2011: Spawn Attempt #4**__

__**6/13/2011: Muk Day**__

Weekly maintenance: tank cleanings, filter changes, etc.

__**6/12/2011**__


 * New employee orientation w/ Beatrice
 * Weekly basement maintenance: water changes, filter changes, etc.
 * Muk checks

__**6/10/2011**__

SAFS GRADUATION =D had the day off

__**6/8/2011: Muk Day**__

Weekly maintenance: tank cleanings, etc.

__**6/3/2011: Laby Extractions**__

media type="custom" key="9796710" width="160" height="160"

__**5/24/2011: Kelp Collection**__

__**5/17/2011:**__ __**Broodstock Genotyping Project - Protocol for HKA Ab Expt #1**__


 * Service the ABI 310:**

After every 5 runs or if machine(s) has not been used in a while. The manual is below for a reference:




 * To prepare ABI 310 buffer solution:**

5 mL 10X ABI 310 stock buffer solution + 45 mL DDI H2O

__50 mL 1X AB 310 buffer solution in a 50 mL conical tube__

__**Prepare 1:60 dilution of each product in new plate:**__

__ 1 uL PCR product __ __ + __ __ 60 uL DDI H2O __

61 uL 1:60 diluted PCR product


 * HKA Ab 1:60 Dilution Plate Map:**


 * || **1** || **2** || **3** || **4** || **5** || **6** || **7** || **8** || **9** || **10** || **11** || **12** ||
 * **A** || HKA43 08:08-1 || HKA43 08:08-2 || HKA43 08:08-3 || HKA43 08:08-4 || HKA43 08:08-5 || HKA43 08:08-6 || HKA43 08:08-7 || HKA43 08:08-8 || HKA56 08:08-1 || HKA56 08:08-2 || HKA56 08:08-3 || HKA56 08:08-4 ||
 * **B** || HKA56 08:08-5 || HKA56 08:08-6 || HKA56 08:08-7 || HKA56 08:08-8 || HKA65 08:08-1 || HKA65 08:08-2 || HKA65 08:08-3 || HKA65 08:08-4 || HKA65 08:08-5 || HKA65 08:08-6 || HKA65 08:08-7 || HKA65 08:08-8 ||
 * **C** || HKA12 08:08-1 || HKA12 08:08-2 || HKA12 08:08-3 || HKA12 08:08-4 || HKA12 08:08-5 || HKA12 08:08-6 || HKA12 08:08-7 || HKA12 08:08-8 ||  ||   ||   ||   ||
 * **D** ||  ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||


 * Formamide/LIZ Master mix:**

__** Per Sample: **__ 10.9 uL Fomamide + 0.4 uL LIZ

11.3 uL master mix per sample

__Mastermix caluclations:__

Formamide: 10.9 uL * 38 =414 uL + Liz: 0.4 uL * 38= 15.2

429.2 master mix


 * Load Plate:**

Pipet 1 uL of 1:60 diluted PCR product + 11.3 uL master mix into each well.

//HKA Ab Expt #1 plate map//
 * || **1** || **2** || **3** || **4** || **5** || **6** || **7** || **8** || **9** || **10** || **11** || **12** ||
 * **A** || HKA43 08:08-1 || HKA43 08:08-2 || HKA43 08:08-3 || HKA43 08:08-4 || HKA43 08:08-5 || HKA43 08:08-6 || HKA43 08:08-7 || HKA43 08:08-8 || HKA56 08:08-1 || HKA56 08:08-2 || HKA56 08:08-3 || HKA56 08:08-4 ||
 * **B** || HKA56 08:08-5 || HKA56 08:08-6 || HKA56 08:08-7 || HKA56 08:08-8 || HKA65 08:08-1 || HKA65 08:08-2 || HKA65 08:08-3 || HKA65 08:08-4 || HKA65 08:08-5 || HKA65 08:08-6 || HKA65 08:08-7 || HKA65 08:08-8 ||
 * **C** || HKA12 08:08-1 || HKA12 08:08-2 || HKA12 08:08-3 || HKA12 08:08-4 || HKA12 08:08-5 || HKA12 08:08-6 || HKA12 08:08-7 || HKA12 08:08-8 ||  ||   ||   ||   ||
 * **D** ||  ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||

Denature plate in thermocycler for 5 mins at 95 deg/
 * Denature product**

Place product in ice bath for ~ 30 seconds out until ready to load
 * Snap cool**


 * Load plate into machine:**
 * Align X-Y-Z axis on machine
 * Place clips on plate
 * Load plate into machine making sure the groove matches up with the plate tray
 * Run plate with following protocols:
 * Module: G5 STR POP 4 (1 mL) g5.md5
 * Matrix File: G5-LIZ, 5-20-02
 * Inj Secs: 5 secs
 * Run Time : 28 mins

__**5/16/2011:**__ __**Broodstock Genotyping Project - PCR**__

Following the same protocol from 5/13/2011 I ran the following two PCRs this morning:

__//Plate 1://__

Run on thermocycler program HKA3 using primers HKA56F_HEX and HKA56R:
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">1 cycle - 10 minutes at 95 degrees
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">30 cycles - 10 seconds at 95 deg, 30 seconds at 56 deg, 45 seconds at 72 deg
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">1 cycle - 10 minutes at 72 deg
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Hold at 4 deg


 * ~  ||~ 1 ||~ 2 ||~ 3 ||~ 4 ||~ 5 ||~ 6 ||~ 7 ||~ 8 ||~ 9 ||~ 10 ||~ 11 ||~ 12 ||
 * ~ A || 10:8-1 ||  ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||
 * ~ B || 10:8-2 ||  ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||
 * ~ C || 10:8-3 ||  ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||
 * ~ D || 10:8-4 ||  ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||
 * ~ E || 10:8-5 ||  ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||
 * ~ F || 10:8-6 ||  ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||
 * ~ G || 10:8-7 ||  ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||
 * ~ H || 10:8-8 ||  ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||

__//Plate 2://__

Run on thermocycler program HKA1 using primers HKA56F_HEX amd HKA56R [column 1] and HKA12F and HKA 12R_HEX:
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">1 cycle - 10 minutes at 95 degrees
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">30 cycles - 10 seconds at 95 deg, 30 seconds at 62 deg, 45 seconds at 72 deg
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">1 cycle - 10 minutes at 72 deg
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Hold at 4 deg


 * ~  ||~ 1 ||~ 2 ||~ 3 ||~ 4 ||~ 5 ||~ 6 ||~ 7 ||~ 8 ||~ 9 ||~ 10 ||~ 11 ||~ 12 ||
 * ~ A || 10:8-1 || 10:8-1 ||  ||   ||   ||   ||   ||   ||   ||   ||   ||   ||
 * ~ B || 10:8-2 || 10:8-2 ||  ||   ||   ||   ||   ||   ||   ||   ||   ||   ||
 * ~ C || 10:8-3 || 10:8-3 ||  ||   ||   ||   ||   ||   ||   ||   ||   ||   ||
 * ~ D || 10:8-4 || 10:8-4 ||  ||   ||   ||   ||   ||   ||   ||   ||   ||   ||
 * ~ E || 10:8-5 || 10:8-5 ||  ||   ||   ||   ||   ||   ||   ||   ||   ||   ||
 * ~ F || 10:8-6 || 10:8-6 ||  ||   ||   ||   ||   ||   ||   ||   ||   ||   ||
 * ~ G || 10:8-7 || 10:8-7 ||  ||   ||   ||   ||   ||   ||   ||   ||   ||   ||
 * ~ H || 10:8-8 || 10:8-8 ||  ||   ||   ||   ||   ||   ||   ||   ||   ||   ||

Lastly, I pipetted 1 uL of each primer PCR product into the individuals corresponding well in column 12 of the second plate to create a 4 uL 1:1:1:1 mixture. Below is the new plate layout for that plate:


 * ~  ||~ 1(//HKA12//) ||~ 2(//HKA65//) ||~ 3 ||~ 4 ||~ 5 ||~ 6 ||~ 7 ||~ 8 ||~ 9 ||~ 10 ||~ 11 ||~ 12(//1:1:1:1//) ||
 * ~ A || 10:8-1 || 10:8-1 ||  ||   ||   ||   ||   ||   ||   ||   ||   || 10:8-1 ||
 * ~ B || 10:8-2 || 10:8-2 ||  ||   ||   ||   ||   ||   ||   ||   ||   || 10:8-2 ||
 * ~ C || 10:8-3 || 10:8-3 ||  ||   ||   ||   ||   ||   ||   ||   ||   || 10:8-3 ||
 * ~ D || 10:8-4 || 10:8-4 ||  ||   ||   ||   ||   ||   ||   ||   ||   || 10:8-4 ||
 * ~ E || 10:8-5 || 10:8-5 ||  ||   ||   ||   ||   ||   ||   ||   ||   || 10:8-5 ||
 * ~ F || 10:8-6 || 10:8-6 ||  ||   ||   ||   ||   ||   ||   ||   ||   || 10:8-6 ||
 * ~ G || 10:8-7 || 10:8-7 ||  ||   ||   ||   ||   ||   ||   ||   ||   || 10:8-7 ||
 * ~ H || 10:8-8 || 10:8-8 ||  ||   ||   ||   ||   ||   ||   ||   ||   || 10:8-8 ||

Figuring out primer dyes and solutions to possible upcoming issues:

What is a dye doesn't work...
 * Allele || Base Pair Range || Dye we already have || New dye to order ||
 * 56 || 91-148 || Hex || Pet ||
 * 65 || 100-200 || Fam || - ||
 * 40 || 110-180 || Tet || Vic ||
 * 48 || 120-220 || Tet || Ned ||
 * 43 || 179-255 || Fam || - ||
 * 12 || 194-363 || Hex || Pet ||

HKA56_PET doesn't work:
 * Fam
 * HKA56
 * HKA43
 * Pet
 * HKA65
 * HKA12
 * Vic
 * HKA40
 * Ned
 * HKA48

HKA65_FAM doesn't work:
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Fam
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">HKA40
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">HKA43
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Pet
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">HKA56
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">HKA12
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Vic
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">HKA65
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Ned
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">HKA48
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">HKA48

HKA40_VIC doesn't work:


 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Fam
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">HKA43
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">HKA65
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Pet
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">HKA56
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">HKA12
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Vic
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">HKA48
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Ned
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">HKA40

HKA48_NED doesn't work:
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Fam
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">HKA43
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">HKA65
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Pet
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">HKA56
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">HKA12
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Vic
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">HKA48
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Ned
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">HKA40

HKA43_FAM doesn't work:
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Fam
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">HKA56
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">HKA43
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Pet
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">HKA40
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">HKA12
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Vic
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">HKA48
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Ned
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">HKA40

HKA12_PET doesn't work:
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Fam
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">HKA56
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">HKA12
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Pet
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">HKA40
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">HKA43
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Vic
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">HKA48
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Ned
 * <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">HKA40

__**5/13/2011:**__ __**Broodstock Genotyping Project - PCR**__

Diluted and aliquoted all primers except TET
 * 90 uL Low TE + 10 uL

Ran PCR using following protocol on extracted DNA from samples 08:8 1-8 (8 rxns total) using HKA43R_6-FAM and HKA43R primers:
 * UV supplies in hood for 5 mins
 * Thaw master mix ingredients while waiting, making sure to only finger vortexx primers
 * Mix master mix with the following ingredients:
 * Sensimix Probe II - 50 uL
 * PCR Water - 25 uL
 * F primer - 5 uL
 * R primer - 5 uL
 * BSA - 5 uL
 * Cover master mix tube with foil and place in dark spot till ready to load into wells
 * UV supplies, plate, and plate rack for 5 mins
 * Thaw extracted DNA, vortex, and spin down
 * Place plate in rack
 * Add 1.0 uL extracted DNA into each well on the **//left//** side of well <-- Note: I added my samples to column 2


 * ~  ||~ 1 ||~ 2 ||~ 3 ||~ 4 ||~ 5 ||~ 6 ||~ 7 ||~ 8 ||~ 9 ||~ 10 ||~ 11 ||~ 12 ||
 * ~ A || tiny bit of master mix || 10:8-1 ||  ||   ||   ||   ||   ||   ||   ||   ||   ||   ||
 * ~ B ||  || 10:8-2 ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||
 * ~ C ||  || 10:8-3 ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||
 * ~ D ||  || 10:8-4 ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||
 * ~ E ||  || 10:8-5 ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||
 * ~ F ||  || 10:8-6 ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||
 * ~ G ||  || 10:8-7 ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||
 * ~ H ||  || 10:8-8 ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||
 * Add 9.0 uL master mix to each well on the **//right//** side of well
 * Place foil over top of plate and hold hot iron on foil for ~5 seconds to adhere foil and plate together
 * Place in balanced salad spinner and spin for 5-10 seconds
 * Place in thermocycler on the HKA-2 setting:
 * 1 cycle - 10 minutes at 95 degrees
 * 30 cycles - 10 seconds at 95 deg, 30 seconds at 54 deg, 45 seconds at 72 deg
 * 1 cycle - 10 minutes at 72 deg
 * Hold at 4 deg
 * Store at 4 deg until ready to further process

__**5/9/2011: Broodstock Genotyping Project practice round**__

Abalone foot tissue extraction practice using KMS's samples

Notes:
 * Number || Accession # || Notes ||
 * 1 || 08:8-1 ||  ||
 * 2 || 08:8-2 || frost on tissue ||
 * 3 || 08:8-3 ||  ||
 * 4 || 08:8-4 ||  ||
 * 5 || 08:8-5 ||  ||
 * 6 || 08:8-6 ||  ||
 * 7 || 08:8-7 || frost on tissue ||
 * 8 || 08:8-8 ||  ||
 * 9 || 08:8-10 ||  ||
 * Blank || Blank ||  ||
 * Followed Qiagen purification of total DNA from animal tissues (spin-column protocol) protocol
 * Incubated tissue at step 2 for 3 hrs

__**5/6/2011: Broodstock Genotyping Project primers ordered**__



Also picked up Qiagen DNeasy Blood & Tissue kit from stores

__**5/5/2011: Muk day**__

Josh and I worked on broodstock maintenance and pit tagging:
 * make sure both disc tag and bee tag are still attached and visible
 * weight and SL measurements
 * gonad ranked
 * excess sponge removed and gave VERY brief FW rinse
 * pit tagged all using IC gel glue

I also worked on cleaning lab tanks in the morning and checking on cage pilot.

__**5/4/2011**__


 * -80 freezer move and thawed old -80
 * Finished packing OA kits and Siri came and picked up

__**5/3/2011: Sick/Muk day**__

Had the stomach flu :-( but went into Muk in the afternoon and did checks and dissected an animal from GHB #13 that has been withering away the last 3 weeks. Also ran into Jon while there and briefly talked about the broodstock genetic testing project.

__**5/2/2011: Freezer day**__

Finished 240 -20 inventory:

media type="scribd" key="54653107" ARG0="key-eaxb429pfhc5nz4wid4" width="640" height="400"

Spent remainder of the day moving and organizing the new -80. Still need to finish mover CAB's stuff to new -80 in order to thaw old one to get new door gasket.

__**5/1/2011: Weekend Muk Checks**__

__**4/29/2011: -20 freezer thaw**__

Spent day thawing out and inventorying Rm. 240 -20 freezer and packing OA kits.

__**4/28/2011: Muk day**__


 * deep cleaned and organized lab
 * checked cage pilot
 * moved dulce and cleaned empty tank

__**4/26/2011: Muk day**__


 * tank cleanings
 * cleaned empty nereo tank

__**4/25/2011: R/P/P 10:12 extractions**__

Followed Qiagen isolation of DNA from stool for pathogen detection other than added only 1/2 inhibit ex tablet


 * = Sample ||= Weight (g) ||= Used all of sample? ||= Notes ||
 * = 10:12-1 ||= 0.092 ||= yes ||= dried out ||
 * = 10:12-2 ||= 0.081 ||= yes ||=  ||
 * = 10:12-3 ||= 0.163 ||= yes ||=  ||
 * = 10:12-4 ||= 0.192 ||= yes ||=  ||
 * = 10:12-5 ||= 0.205 ||= yes ||=  ||
 * = 10:12-6 ||= 0.137 ||= yes ||=  ||
 * = 10:12-9 ||= 0.024 ||= yes ||= dried out ||
 * = 10:12-10 ||= 0.137 ||= yes ||=  ||
 * = 10:12-11 ||= 0.048 ||= yes ||= dried out ||
 * = 10:12-12 ||= 0.044 ||= yes ||=  ||
 * = 10:12-13 ||= 0.046 ||= yes ||=  ||
 * = 10:12-14 ||= 0.117 ||= yes ||= dried out ||
 * = 10:12-15 ||= 0.132 ||= yes ||=  ||
 * = 10:12-16 ||= 0.020 ||= yes ||= dried out ||
 * = 10:12-17 ||= 0.030 ||= yes ||= dried out ||
 * = 10:12-18 ||= 0.022 ||= yes ||= dried out ||
 * = blank ||= blank ||= blank ||=  ||

//Planning Notes for future qPCR://


 * ~  ||~ 1 ||~ 2 ||~ 3 ||~ 4 ||~ 5 ||~ 6 ||~ 7 ||~ 8 ||~ 9 ||~ 10 ||~ 11 ||~ 12 ||
 * ~ A || 10:7-1 || 10:7-1 || 10:7-10 || NTC || 10:11-7 || 10:11-7 || 10:11-15 || 10:11-15 || 10:11-23 || 10:11-23 || std-1 || std-1 ||
 * ~ B || 10:7-2 || 10:7-2 || 10:7-10 || NTC || 10:11-8 || 10:11-8 || 10:11-16 || 10:11-16 || 10:11-24 || 10:11-24 || std-2 || std-2 ||
 * ~ C || 10:7-3 || 10:7-3 || 10:11-1 || 10:11-1 || 10:11-9 || 10:11-9 || 10:11-17 || 10:11-17 || 10:11-25 || 10:11-25 || std-3 || std-3 ||
 * ~ D || 10:7-4 || 10:7-4 || 10:11-2 || 10:11-2 || 10:11-10 || 10:11-10 || 10:11-18 || 10:11-18 || 10:11-26 || 10:11-26 || std-4 || std-4 ||
 * ~ E || 10:7-5 || 10:7-5 || 10:11-3 || 10:11-3 || 10:11-11 || 10:11-11 || 10:11-19 || 10:11-19 || 10:11-27 || 10:11-27 || std-5 || std-5 ||
 * ~ F || 10:7-7 || 10:7-7 || 10:11-4 || 10:11-4 || 10:11-12 || 10:11-12 || 10:11-20 || 10:11-20 || 10:7-B || 10:7-B || std-6 || std-6 ||
 * ~ G || 10:7-8 || 10:7-8 || 10:11-5 || 10:11-5 || 10:11-13 || 10:11-13 || 10:11-21 || 10:11-21 || 10:11-B || 10:11-B || std-7 || std-7 ||
 * ~ H || 10:7-9 || 10:7-9 || 10:11-6 || 10:11-6 || 10:11-14 || 10:11-14 || 10:11-22 || 10:11-22 || 10:12-B || 10:12-B || std-8 || std-8 ||


 * ~  ||~ 1 ||~ 2 ||~ 3 ||~ 4 ||~ 5 ||~ 6 ||~ 7 ||~ 8 ||~ 9 ||~ 10 ||~ 11 ||~ 12 ||
 * ~ A || 10:11-28 || 10:11-28 ||  || 10:7-11 || 10:7-11 || 10:12-4 || 10:12-4 || 10:12-14 || 10:12-14 ||   || std-1 || std-1 ||
 * ~ B || 10:11-29 || 10:11-29 ||  || 10:7-12 || 10:7-12 || 10:12-5 || 10:12-5 || 10:12-15 || 10:12-15 ||   || std-2 || std-2 ||
 * ~ C || 10:11-30 || 10:11-30 ||  || 10:7-13 || 10:7-13 || 10:12-6 || 10:12-6 || 10:12-16 || 10:12-16 ||   || std-3 || std-3 ||
 * ~ D || 10:11-31 || 10:11-31 ||  || 10:7-14 || 10:7-14 || 10:12-9 || 10:12-9 || 10:12-17 || 10:12-17 ||   || std-4 || std-4 ||
 * ~ E || 10:11-32 || 10:11-32 ||  || 10:7-15 || 10:7-15 || 10:12-10 || 10:12-10 || 10:12-18 || 10:12-18 ||   || std-5 || std-5 ||
 * ~ F ||  ||   ||   || 10:12-1 || 10:12-1 || 10:12-11 || 10:12-11 ||   ||   ||   || std-6 || std-6 ||
 * ~ G ||  ||   ||   || 10:12-2 || 10:12-2 || 10:12-12 || 10:12-12 || NTC ||   ||   || std-7 || std-7 ||
 * ~ H ||  ||   ||   || 10:12-3 || 10:12-3 || 10:12-13 || 10:12-13 || NTC ||   ||   || std-8 || std-8 ||
 * 1) of reactions:

Samples-65*2=130 Negative-2*2=4 Standards-8*2*2=32 Pipet Error-166*10%~16

TOTAL RXN's = 182

Reagent Master Mix amounts:

Immo- 12.5*182= 2275 uL BSA- 1.5*182= 273 uL WSN-F 0.8*182= 145.6 uL WSN-R 0.8*182= 145.6 uL Probe 0.5*182= 91 uL H2O 6.9*182= 1255.8 uL

Double check: All reagents added up/# samples = 4186/182 = 23 uL

__**4/19/2011**__

__Proposed Methods for Z. marina (taken and modified from Craven et al 2005):__

//Isolation:// Isolates will be provided from FHL. I believe 4 isolates are ready and will be brought back tomorrow.

//Agar//: Following Porter 1987, all cultures to be maintained on 1% serum seawater agar. media type="custom" key="9147058"

//DNA isolation:// Grow cultures for 4 days on solid media. Harvest cells by cutting agar blocks into sterile deionized water. Provide vigorous agitation to liberate cells followed by filtration to remove agar blocks. Centrifuge at 13,000 RPM for 5 minutes.

//DNA extraction:// Will follow the microwave miniprep method (Goodwin and Lee 1993) <--will run over to HSL this afternoon to get protocol

//PCR:// **need to discuss primers still**


 * PCR rxn will be 30uL containing:
 * 15mM Tris-HCL
 * 1.5mM MgCl2
 * 50mM KCl
 * 200 uM of each deoxynucleotide triphosphate (dATP, dCTP, dGTP, dTTP) [Panvera]
 * 200 nM of primers
 * 0.025 U uL-1 Taq DNA polymerase [Qiagen]
 * 10 ng genomic DNA (water for negative control)
 * use Nanodrop
 * If need be, use (C1)(V1)=(C2)(V2), to normalize samples by calculating how much concentrated genomic DNA is needed to make a 500 µ L of 10 ng/ µ L genomic DNA stock from our initial concentrations)
 * Make sure A260/A280 ratios falls between 1.7 and 2.0 for “pure” DNA
 * Thermal cycler program:
 * 30 cycles of:
 * 1 min @ 95 C
 * 1 min @ 55 C
 * 1 min @ 72 C
 * 5 min at 72 C
 * Electrophoresis (visualization)
 * 0.8% agarose gel electrophoresis
 * Mix 1.2g of agarose with 150mL 1x TAE in a 1L flask
 * Microwave solution for ~ 3 minutes swirling occasionally
 * Cool solutionand add 15 uL ethidium bromide (EtBr) and mix by swirling
 * Pour into gel tray, add gel combs, and allow to set and cool
 * Estimate concentration of samples using 100 bp ladder [Panvera]
 * Clean PCR products
 * Using <span style="font-family: arial,sans-serif; font-size: 11px; line-height: normal; white-space: nowrap;">QIAquick PCR Purification Kit (protocol: http://kirschner.med.harvard.edu/files/protocols/QIAGEN_QIAquickSpin_EN.pdf)
 * Using <span style="font-family: arial,sans-serif; font-size: 11px; line-height: normal; white-space: nowrap;">QIAquick PCR Purification Kit (protocol: http://kirschner.med.harvard.edu/files/protocols/QIAGEN_QIAquickSpin_EN.pdf)

//Sequence and Analyze//


 * __4/13/2011__**


 * Packed up histo chemicals for Muk
 * Sent out an email of basement reminders
 * Typed up lab meeting notes and forwarded to all lab members, including a tutorial for logging on to the wiki
 * Forwarded email about WS suspension to Brent
 * Emailed Sandy about //Z. marina// project
 * Filter changes, checks, and basement clean up
 * Printed off a hard copy of all lab members notebooks
 * Started new ordering book binder for 2011
 * Updated wiki page with new pages
 * Printed off trouble shooting guide for gel box power supplies


 * __4/12/2011__**

I spent the morning fixing the gel box power supplies. We then had our lab meeting and following that I shadowed Sam for a little bit catching up on the progress of the cloning. I then went up to Mukilteo to check in on the abalone since Josh is in the field for the outplant.


 * __4/11/2011: Mukilteo Day__**

Today was the last outplant day. I spent the morning helping load up the abalone into outplant tubes and into the WDFW truck. Once everybody headed up north, I stuck behind and cleaned up GH B. I split the dulce culture into two tanks to help promote growth. I also combined the nereo into one tanks and then cleaned out the two empty algae tanks. Once Jessie got their, I helped record her data for the PIT tagging project as she scanned the abalone for tags.


 * __4/10/2011: Weekend Muk Checks__**


 * __4/8/2011__**

I spent the morning hanging out in the basement doing checks, feeding the abalone, finishing setting the tote systems back up, and finishing cleaning the basement up. After that I made 1 L of the following filter sterilized preservation solution for Carolyn: 0.5M NaCl (29.2215 g/L), 10mM Tris-HCl (1.5756 g/L), and 100 mM EDTA (37.224 g/L). I also filled up ~40 cryovials with 1 mL RNA later in each and autoclaved some 2 mL eppendorf tubes for Carolyn.


 * __4/7/2011__**

Today was a Muk day. I cleaned and fed all tanks other than the post-set tanks that were done earlier in the week. I also changed all the filters in the lab and finished cleaning some PVC standpipes and post-set plates.


 * __4/6/2011__**

I spent the morning doing filter changes and organizing the basement. I started resetting up systems for the antibiotic trial as well. After that, I shadowed Sam over in the Roberts Lab. We were supposed to start cloning today, but his scheduled changed a bit so he was working on finishing up a RACE for cyclooxygenase (brief notes: causes inflammation, found that there are two isomers in oysters, they know 1 but are working on the 2nd) for both the 5' and 3' directions when I got there and then set up a PCR reaction using his RACE products for both. After that we talked through the cloning protocol which he is going he is going to start tomorrow while I'm at Muk and we will regroup on Friday when I'm back in the lab.

Just for my memories sake I did a little research on both cloning and RACE to help me better understand the usefulness and techniques of both. Below is what I found:

__RACE:__

media type="custom" key="8994570"

media type="custom" key="8994700"

__Cloning:__

Roberts Lab Cloning Protocol

media type="youtube" key="KQNyxwzBnjw" height="234" width="288"

__**4/5/2011: R/P/P 10:11 extractions continued**__

Followed Qiagen isolation of DNA from stool for pathogen detection other than added only 1/2 inhibit ex tablet


 * = Sample ||= Weight (g) ||= Used all of sample? ||= Notes ||= Round ||
 * = 10:11-17 ||= 0.057 ||= yes ||=  ||= 1 ||
 * = 10:11-18 ||= 0.085 ||= yes ||=  ||= 1 ||
 * = 10:11-19 ||= 0.062 ||= yes ||=  ||= 1 ||
 * = 10:11-20 ||= 0.043 ||= yes ||=  ||= 1 ||
 * = 10:11-21 ||= 0.081 ||= yes ||=  ||= 1 ||
 * = 10:11-22 ||= 0.115 ||= yes ||=  ||= 1 ||
 * = 10:11-23 ||= 0.124 ||= yes ||=  ||= 1 ||
 * = 10:11-24 ||= 0.096 ||= yes ||=  ||= 1 ||
 * = 10:11-25 ||= 0.066 ||= yes ||=  ||= 2 ||
 * = 10:11-26 ||= 0.041 ||= yes ||=  ||= 2 ||
 * = 10:11-27 ||= 0.042 ||= yes ||=  ||= 2 ||
 * = 10:11-28 ||= 0.194 ||= yes ||=  ||= 2 ||
 * = 10:11-29 ||= 0.083 ||= yes ||=  ||= 2 ||
 * = 10:11-30 ||= 0.092 ||= yes ||=  ||= 2 ||
 * = 10:11-31 ||= 0.012 ||= yes ||= dried out ||= 2 ||
 * = 10:11-32 ||= 0.049 ||= yes ||=  ||= 2 ||
 * = blank ||= blank ||= blank ||=  ||= 2 ||

__**4/4/2011: R/P/P 10:11 extractions**__

Followed Qiagen isolation of DNA from stool for pathogen detection other than added only 1/2 inhibit ex tablet


 * = Sample ||= Weight (g) ||= Used all of sample? ||= Notes ||= Round ||
 * = 10:11-1 ||= 0.115 ||= yes ||=  ||= 1 ||
 * = 10:11-2 ||= 0.158 ||= yes ||=  ||= 1 ||
 * = 10:11-3 ||= 0.072 ||= yes ||=  ||= 1 ||
 * = 10:11-4 ||= 0.102 ||= yes ||=  ||= 1 ||
 * = 10:11-5 ||= 0.083 ||= yes ||=  ||= 1 ||
 * = 10:11-6 ||= 0.200 ||= yes ||=  ||= 1 ||
 * = 10:11-7 ||= 0.103 ||= yes ||=  ||= 1 ||
 * = 10:11-8 ||= 0.050 ||= yes ||= dried out ||= 1 ||
 * = 10:11-9 ||= 0.150 ||= yes ||=  ||= 2 ||
 * = 10:11-10 ||= 0.100 ||= yes ||= dried out ||= 2 ||
 * = 10:11-11 ||= 0.158 ||= yes ||=  ||= 2 ||
 * = 10:11-12 ||= 0.043 ||= yes ||= dried out ||= 2 ||
 * = 10:11-13 ||= 0.101 ||= yes ||=  ||= 2 ||
 * = 10:11-14 ||= 0.152 ||= yes ||=  ||= 2 ||
 * = 10:11-15 ||= 0.055 ||= yes ||= dried out ||= 2 ||
 * = 10:11-16 ||= 0.064 ||= yes ||= dried out ||= 2 ||

__**3/30/2011 & 4/1/2011: Friedman Lab Wiki Page**__

I worked on setting up the new wiki page for our lab. I also did routine basement checks and weekly filter changes.

__**3/31/2011: Mukilteo Day**__

I cleaned and fed all the tanks in the hatchery. I also cleaned three large algae tanks in preparation of Josh bringing back some yummy nereo for the abalone that he collected while out in the field for the outplant.

__**3/29/2011: Mukilteo/Outplant Day**__

Abalone outplant day =) With the help of the WDFW dive team and PSRF folks we loaded up abalone from 4 of the 6 outplant sites to move them up north. We also had guests from NOAA and KOMO filming us prepping the abalone for their epic journey into the wild. After all the animals were loaded into the fish totes in the back of the two trucks and everyone left to go up north, I stayed back and cleaned up GH B and then scrubbed the now 4 empty tanks clean for future abalone.

__**3/28/2011 @ 4:00 pm: Daily Update**__

__Lab maintenance__: I'm still chugging along at filing all our slides into an orderly fashion into our new slide drawers we got in =). Between last Friday and today I'm about 1/3 of the way done. I hope to finish by the end of the week. Once we get histo block drawers in I will conquer that hurdle as well in hopes to have everything in easy access for reference, screening, etc. to save time.

__OA outreach:__ My talk at Ballard HS went very well today. I made sure the students had enough time for Q&A's in the beginning for general OA information they were still curious about. I then asked for general hypotheses from the class as what they expect to happen to grey scale values, pH, and buoyant weight. We then finished setting up there experiments, which included making their carbon factories, adding seawater, taking initial measurements, and introducing the spat to their systems. I wished we would have had enough time to cover oyster life history but we ran out of time. I did briefly explain how oysters deposit new shell and that their leading edge is very fragile, especially when exposed to acidified conditions. Overall, the students seemed really excited for the experiment and it was fun to watch them learn experimental set-up.

__//Zostera marina// project__: I started looking at NCBI for primers for this upcoming project of mine and came across two sequences that are below for :

<span class="wiki_link_ext">GenBank: AF265335.1 []

GenBank: AF265334.1 []

I designed primers for the <span class="wiki_link_ext">AF265335.1 (the top sequence) with a max temp diff of 2 deg C, size 18-30 w/ optimum at 20, GC clamp at end of 3' at 1, and minimal GC percentage. My top two picks are:

Primer pair 9
Products on intended target
 * ~  ||~ Sequence (5'->3') ||~ Strand on template ||~ Length ||~ Start ||~ Stop ||~ Tm ||~ GC% ||
 * ~ Forward primer || AGCCAAGTCTGGTGCCAGCAG || Plus || 21 || 587 || 607 || 59.31 || 61.90% ||
 * ~ Reverse primer || TTTTCAGCCTTGTGACCATATTCCCC || Minus || 26 || 1157 || 1132 || 57.36 || 46.15% ||
 * ~ Internal oligo ||  || Plus ||   ||   ||   ||   ||   ||
 * ~ Product length |||||||||||| 571 ||
 * ~ Product Tm ||||||||||||  ||
 * ~ Product Tm - min(OLIGO Tm) ||||||||||||  ||
 * ~ Exon junction ||||||||||||||||  ||
 * ~ Total intron size ||||||||||||||||  ||

>AF265335.1 Labyrinthula zosterae type-154 small subunit ribosomal RNA gene, partial sequence code product length = 571 Forward primer 1 AGCCAAGTCTGGTGCCAGCAG 21 Template 587 ..................... 607

Reverse primer 1 TTTTCAGCCTTGTGACCATATTCCCC 26 Template 1157 .......................... 1132

Primer pair 7
Products on intended target
 * ~  ||~ Sequence (5'->3') ||~ Strand on template ||~ Length ||~ Start ||~ Stop ||~ Tm ||~ GC% ||
 * ~ Forward primer || GGAGCCAAGTCTGGTGCCAGC || Plus || 21 || 585 || 605 || 59.98 || 66.67% ||
 * ~ Reverse primer || CCCGGAACCCAAAAACTTTGATTTCTCG || Minus || 28 || 1132 || 1105 || 59.12 || 46.43% ||
 * ~ Internal oligo ||  || Plus ||   ||   ||   ||   ||   ||
 * ~ Product length |||||||||||| 548 ||
 * ~ Product Tm ||||||||||||  ||
 * ~ Product Tm - min(OLIGO Tm) ||||||||||||  ||
 * ~ Exon junction ||||||||||||||||  ||
 * ~ Total intron size ||||||||||||||||  ||

>AF265335.1 Labyrinthula zosterae type-154 small subunit ribosomal RNA gene, partial sequence code product length = 548 Forward primer 1 GGAGCCAAGTCTGGTGCCAGC 21 Template 585 ..................... 605

Reverse primer 1 CCCGGAACCCAAAAACTTTGATTTCTCG 28 Template 1132 ............................ 1105

__**3/25/2011: Slide Organization**__

I worked on organizing and filing slides all day.


 * __3/24/2011: Mukilteo Day__**

__**3/23/2011**__

I picked up the OA kits from Seattle Academy then changed filters in the basement. I restocked two OA kits for Siri to pick up on Friday to deliver to Ballard HS. I spent the remainder of the day putting together an order sheet for Robyn for the SOC abalone broodstock conditioning feed.

media type="custom" key="8975184" width="180" height="180"


 * __3/22/2011: Mukilteo Day__**


 * __3/21/2011__**

I worked on water filtering today for Lisa. I also ran through the pH protocol using the new spec machine for Elene.


 * __3/20/2011: Weekend Muk Checks__**


 * __3/18/2011__**

I worked on rm 008 ordering supply list for the items that need to be replaced from our Cu experiment from FISH 441. I also worked on water filtering for Lisa and started researching feed supply info for the SOC broodstock feed.

media type="custom" key="8975196" width="200" height="200"