2B-RAD Protocol

2b-RAD protocol

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Wednesday 8/12/15- Friday 8/14/15

Wednesday 8/12/15

Larvae tanks

  • NF_Tank2_160 (224) -> NF cultch set
  • NF_Tank2_160 (100) -> swimmers only
  • HC_Tank2_160 (224) -> HC cultch set
  • HC_Tank2_160 (100) -> swimmers only
  • SS_Tank2_160 (224) -> barely any, put back in tank
  • SS_Tank2_160 (100) -> swimmers only

Tile Set

  • Did live/dead counts for both A and B
  • Did not add the unset larvae back to A
  • Rinsed tiles

Cultch Set

  • NF >450 -> NF >1000
  • SS > 450 -> SS>1000
  • SS_SetA -> SS > 450; SS_SetA

Thursday 8/13/15

Started dissections of tissue from the broodstock for subsequent DNA extractions with the help of Brent Vadopalas and the PSRF intern Ryann. First, we recorded the weight of each oyster in a family and placed it on a numbered pad.

Broodstock dissection setup

Broodstock dissection setup

A picture was taken of the entire family with a ruler for later measurement in ImageJ. Effort was made to hold the phone level to avoid the impact of tilt on apparent size. We made a little assembly line, with Brent shucking before joining Ryann and I in dissecting out adductor muscle tissue and storing in 1.5 mL tubes with 1 – .75 mL RNALater. If there was not very much muscle tissue, mantle or the entire oyster were taken as well. Scalpels and forceps were rinsed sequentially in soapy water, bleach, and freshwater between each oyster. Fresh scalpels were used between populations. We got through SS2, SS1,SS3, SS4, SS5, and NF4, averaging about 45 minutes per family by the time we got the hang of it.

Husbandry

  • Rinsed tiles and cultch
  • Feeding

Friday 8/14/15

Larvae tanks

  • NF_Tank2_160 (224) -> NF_New cultch set
  • NF_Tank2_160 (100) -> dumped
  • HC_Tank2_160 (224) -> none
  • HC_Tank2_160 (100) -> dumped
  • SS_Tank2_160 (all) -> dumped

Tile Set Counts

  • NF_SetA
  • SS_SetA
  • HC_SetA
  • HC_SetB
  • SS_SetB

Dissections

Did NF2 and NF1 with a little help from Ryann on NF1

Thursday 7/30/15 (Lab work!)

I got to do some bona fide lab work today, which was a nice change of pace. I’ve been taking samples of larvae for DNA sequencing at various points throughout the experiment:

  • From all newly released larvae (either from each family or combined, depending on how I filtered them out)
  • From larvae in the “New” tanks that reach 160 microns
  • From larvae in the “160” tanks that reach 224 in size
  • Occasionally pooled larvae from a tank

These samples have mostly been stored at -20degC in .5-1 mL of RNALater, but duplicates of many were also stored in ethanol (1st in 75%, then in 95%). Earlier in the summer I wanted to do a test extraction to see if there was a particular storage method that worked best and figure out which extraction kit to use, but then the oysters needed maintenance 6 days a week and all of a sudden in was almost August. With the growth rate experiments and larval production essentially done, I finally had a day to do the test extraction.

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Protocol for 16S PCR, SAP/Exo, Sequencing Reaction

Dilute primers to 100 uM by adding 10x # of nmol H2O
Working stock (5 uM): 2.5uL stock, 47.5uL dH2O
PCR
MM1
Comp
1x Volume
40x
Final Conc
Water
6 μl
240
dNTPs
.5 μl
20
200 μM
R primer
2.5  μl
100
0.5 μM
F primer
2.5 μl
100
0.5 μl
MM2
Water
9.17 μl
366.8
10x buffer
3.2 μl
128
2 mL MgCl2
Taq
.13 μl
5.2
1.25 U/rxn
Add 1 μl DNA to each tube
Step
Cycles
Time
Temp
Initial Denaturation
1
2 min
94 deg
Denaturation
Annealing
Elongation
1-8
30 s
1 min
1 min
94
54,53,52,51,50,49,48,48
72
Denaturation
Annealing
Elongation
9-33
30 s
1
1
94
48
72
Final Elongation
1
7
72
forever
10
SAP/Exo
MM
Reagent
1x
13x
Sap
0.5 ul
6.5
Exo
0.5 ul
6.5
5 ul of PCR product + 1uL MM
Step
Time
Temp
Incubate
30 min
37 C
Denature
15 min
80 C
Chill
forever
25 C
Sequencing