Wednesday 11/25/15

Ran gels of samples B4-B10 from 2bRAD Library 1 of the PCR done on 11/24/15. Some of the PCRs either did not seem to work or might’ve washed out of the gel. I still cut out bands around 170bp for these, but will look at the quantification to see if anything is recovered. Gel slices were left in 4degC.

Samples that did not work (likely need redos): F5 8(HC1_4B), E5 (HC1_9), B5(HC2_13), B7 (HC3_9), B8(HC3_17, library 4).

 

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Tuesday 11/24/15

Finished gel extraction of samples B3-C4 from 2bRAD Library 1 started on 11/23/15 following the protocol described in that post.

Did the PCR of the rest of Library 1 (B5-C12) with the new Taq. There is no H9, however (due to how I labelled on the spreadsheet).

First made more Lib 1 and Lib 2 primer stock:

  • 10 uL stock + 90 uL NFW = 10 uM

PCR master mix. Added 3.75 of each 1 uM barcode (HT and BC) to wells.

1x
65
10 mM (each) dNTPS
1.5
97.5
10 uM ILL-Lib1
1.5
97.5
10 uM ILL-Lib2
1.5
97.5
5X Q5 buffer
15
975
Q5 Taq polymerase
.75
48.75
20.25 + barcode = 27.75
648

 

Monday 11/23/15

Took the tubes with gel slices from Sunday 11/22/15 and centrifuged them at high speed for 1 minute to bring gel in contact with water. Put them in the -80degC freezer for 1.5 hours (Meyer protocol recommends at least 1 hour). Afterwards, centrifuged at maximum speed in the refrigerated centrifuge at 4degC for 15 minutes (Meyer protocol recommends 10-20 minutes). Then pressed gel slice against tube wall and took out between 30-50uL per sample and added then to a PCR plate corresponding to their well positions during the previous 2bRAD steps. I found that if the gel slices were left out of the cold for too long that they were more likely to break up during this step and less supernatant was recovered- in the future will work in batches of 16 and leave the rest in the fridge. Leftover gel slices were put back in the fridge in case they needed to be gel extracted using a kit due to low DNA recovery.

Ran out the gel of wells B3-C4 on a regular gel and cut out the band at around 170bp. Still need to do B4 from the PCR on 11/22/15. Left gel in 40 uL overnight at 4degC.

Gel_2bRAD_Lib1_B3_C4

Gel of samples B-C4 of 2bRAD Library 1

Did the Ligation 2bRAD step on libraries 2 and 3- decided to use 4 as a backup for failed individuals and put that plate in the freezer.

First made new adapters by adding the oligos into 2 separate tubes and leaving at room temp for 10 minutes:

Adaptor 1
150x 
5ILL-NR
7.5
Anti-ILL
7.5
NFW
735
Adaptor 2
150x
3ILL -NR
7.5 uL
Anti-ILL
7.5 uL
NFW
735 uL
Ligation master mix.
1x
149x
2 uM Adaptor 1
5 uL
745
2 uM Adaptor 2
5 uL
745
T4 ligase
1 uL
149
T4 ligase buffer with 10 mM ATP
4 uL
596
NFW
22
3278
10 mM ATP
1
149
Total
38
5662

Added 38 uL to each well.

11/21/15 and 11/22/15

Saturday 11/21/15

Added 8 uL of nuclease free water to Libraries 2, 3, and 4. Put in 4degC.

Sunday 11/22/15

Set up a PCR of wells H1-B4 of Library 1 (did not have enough Taq to do any more so ordered more Q5 High-Fidelity Taq). As the 100 uL PCR reactions recommended in the Meyer 2bRAD protocol always overfill my gel wells, I rescaled the recipe to be 77.75 uL reactions (50 uL ligation product + 3.75uL each barcode + 20.25 uL master mix).

First, made new working stocks of primers:

  • Made 10 uM stock of Lib 1 and Lib 2
    • 5 uL stock + 45 uL NFW
  • Made 1 uM stock of BC11, BC12, HT2, HT3, HT4, HT5, HT6, HT7, HT8
    • 1 sock + 99 NFW
1x
32
10 mM (each) dNTPS
2: 1.5
48
10 uM ILL-Lib1
2: 1.5
48
10 uM ILL-Lib2
2: 1.5
48
5X Q5 buffer
20: 15
480
Q5 Taq polymerase
1: .75
24
20.25
648

Added 3.75 uL of each barcode (this took a lot of time. I marked on the datasheet as I went to make sure I added barcodes to each well.)

Made a large gel with 24 wells and a regular gel with 8 wells. The top part of the large well broke so was only able to run 22 of the 31 PCRs (H1-C3). I put the gel slices in 1.5mL tubes with 40 uL of NFW and left overnight in the fridge.

Did the AlfI digestion of libraries 2, 3, and 4.

1x
230x
10x buffer R
1.2 uL
276
150 uM SAM
.8
184
AlfI (2 U/uL)
.5
115
water
1.5
345
4
920

Thermocycler at 37degC for 2 hours and 65degC for 15 minutes. Put in fridge afterwards.

11/19/15

DNA extraction of common garden broodstock:

  1. SS4_2b (redo of extraction attempted on 11/18/15)
  2. NF5_1
  3. NF5_2
  4. NF5_3
  5. NF5_4
  6. NF5_5
  7. NF5_6
  8. NF5_7
  9. NF5_8
  10. NF5_9
  11. NF5_10
  12. NF5_11
  13. NF5_12
  14. NF5_13
  15. NF5_14
  16. NF5_15
  17. NF5_16
  18. NF5_17
  19. NF5_18
  20. NF5_19

Afterwards, got the concentration of these extractions with Qubit. Plated of 1ug of DNA for Libraries 2, 3, and 4 (see “Libraries” sheet of the Common Garden Samples datasheet). Put Airpore tape over the plates and left in an incubator at 37degC.

11/18/15

DNA extraction of common garden broodstock. Eluted in 200 uL.

  1. NF3_17
  2. NF3_18
  3. NF3_19
  4. NF3_20
  5. NF4_1
  6. NF4_2
  7. NF4_3
  8. NF4_4
  9. NF4_5
  10. NF4_6
  11. NF4_7
  12. NF4_8
  13. NF4_9
  14. NF4_10
  15. NF4_11
  16. NF4_12
  17. NF4_13
  18. NF4_14
  19. NF4_15
  20. NF4_16
  21. NF4_17
  22. NF4_18
  23. NF4_19
  24. NF5_1
Did the ligation step for Plate 1 of the 2b-RAD broodstock libraries.
1st made fresh annealed adaptors.
Adaptor 1
96x 
5ILL-NR
5
Anti-ILL
5
NFW
490
Adaptor 2
11x
3ILL -NR
5 uL
Anti-ILL
5 uL
NFW
490 uL
Removed 5 uL of digestion product from each well to give 10 uL.
1x
96x
2 uM Adaptor 1
5 uL
480
1 uM Adaptor 2
5 uL
480
T4 ligase
1 uL
96
T4 ligase buffer with 10 mM ATP
4 uL
384
NFW
24
2304
10 mM ATP
1
96
Total
40
3840

Let to digest at 16degC for 2 hours followed by 10 minutes at 62degC and then put in freezer.

In the evening did another set of extractions. These are repeats of the ones done on 10/27/15 that turned out poorly.

  1. SS4_1
  2. SS4_2 (messed up)
  3. SS4_3
  4. SS4_4
  5. SS4_5
  6. SS4_6
  7. SS4_7
  8. SS4_8
  9. SS4_9
  10. SS4_10
  11. SS4_11
  12. SS4_12
  13. SS4_13
  14. SS4_14
  15. SS4_15
  16. SS4_16
  17. SS4_17
  18. SS4_18

When vortexing SS4_2 the cap wasn’t on all the way so most of it splashed out. Need to re-extract.

 

11/16/15 and 11/17/15

Monday 11/16/15

Took out the plate  from the incubator that was setup on Friday 11/13/15. The samples were completely dried down. I added 10 uL NFW to each well and left the plate in the fridge overnight. I also Qubit-ed more DNA extractions.

Tuesday 11/17/15

DNA extraction of broodstock from common garden experiment.

  1. NF1_9
  2. NF1_10
  3. NF1_11
  4. NF1_12
  5. NF1_13
  6. NF1_14
  7. NF1_15
  8. NF1_16
  9. NF1_17
  10. NF1_18
  11. NF1_19
  12. NF1_20
  13. NF1_21
  14. NF3_6
  15. NF3_7
  16. NF3_8
  17. NF3_9
  18. NF3_10
  19. NF3_11
  20. NF3_12
  21. NF3_13
  22. NF3_14
  23. NF3_15
  24. NF3_16

Eluted these in 200 uL to increase yield. Used Qubit to get concentration.

Set up a digestion of the 1st 2bRAD library plate. To save time, I did not take out 2 uL from each well to make 8uL as listed in the protocol. Instead I increased all of the master mix reagents so they would add up to 5uL, therefore maintaining a 2:1 sample:master mix ratio.

1x
96x
10x buffer R
1.2 uL -> 1.5
144
150 uM SAM
.8 -> 1
96
AlfI (2 U/uL)
.5 -> .625
60
water
1.5 -> 1.875
180
Total uL
5

Let digest for 2 hours at 37degC followed by 15 minutes at 65degC then put in the fridge.

Friday 11/13/15

Starting to setup the 2b-rad libraries of adult broodstock that will be sent off in a couple weeks for sequencing. I made a sheet labelled “Libraries” on the Common Garden DNA Samples datasheet outlining which sample will go in which library. With our barcodes we can sequence up to 96 samples per lane. I decided to do about 75 per lane across 4 lanes, mixing up the 3 populations across the lanes. I’ll also repeat 3 samples across all lanes and repeat 2 samples within each lane.

I got the concentration of the extractions for NF2 with Qubit and then pipetted out 1 ug of DNA for the samples in Library 1 in a 96 well plate. I put Airpore tape over the plate and left it in a 37degC incubator to dry out.

 

Friday 11/6/15

At 9:00am I turned off the chiller and took a sample of oysters from the overnight treatment. It appeared that many of the oysters from the overnight treatment were gaping slightly, but they still offered resistance during dissection. I dissected 6 cultch set oysters per population and 4 tile set oysters and put the whole body tissue in RNALater. I also dissected 10 oysters per population from the control treatment. Instead of moving the overnight oysters over to the control tank, I let the temperature gradually increase by draining some of the tank and adding ambient water. We switched the control tank over to a flow-through system with live algae dripping in. At 2:00pm I moved the overnight chilled oysters to the control tank. The hatchery folks are going to keep an eye on the oysters for 2 weeks, after which they’ll sample all of the survivors.

Time
9:00am
-control tank: 9degC
-chilled tank: 0degC; turned off chiller
10:30am
– chilled tank: 3degC
12:00pm
-chilled tank: 5degC
1:00pm
-chilled tank: 7degC
-control tank: 10degC; switched to flowthrough system
2:00pm
-chilled tank: 9degC; added overnight oysters to control tank
-control tank: 11degC

I took apart the tile trays and drained out the tank they had been in. Some tiles that had only a few oysters on them were not used in the temperature experiment. I dissected these and put the whole body in RNALater, figuring they could be used for genotyping or looking at epigenetic in the F2 generation. Some of them I took a picture for size data and some of them I measured with a ruler.

Samples from tiles:

  1. SSB10
  2. NFA7_1
  3. NFA7_2
  4. SSB11
  5. NFA13R: 1.3cm x 1.4cm
  6. HCA10_1: 1.3 x 1.2
  7. HCA10_2: .9 x .7
  8. HCA15 (top)
  9. HCA6 (top)
  10. NFB12 (middle)
  11. NFB13R (2nd from top)
  12. NFB7 (left)
  13. HCA8

Wednesday 11/4/15 and Thursday 11/5/15

Wednesday 11/4/15

Gave a talk to the PSRF monthly meeting in the morning. Afterwards I went with Alice to the dock and brought up the cultch set F2 oysters to the hatchery. As there weren’t very many tile set oysters, I wanted to supplement the experiment with these. I put them in one of the setting tanks outside in static, ambient water.

Thursday 11/5/15 (Experiment Day)

Alice and I built a little “house” for the chiller to protect it from the rain. We set up the chiller in the smaller section of the setting tank in order to cool down the water quicker. The chiller was set to 0degC and turned on at 10am. Water on the other side was at 11degC, a little cooler than ambient due to the air temperature overnight. For the controls, 250+/- 5 cultch set oysters from each population were placed in 180 micron silos hanging in the ambient temperature side of the tank. I put around 350 oysters in silos on the chilled side at 11:30am when the temperature was at 7degC. At 1:00pm the temperature was at 0degC and maintained that temperature all day. At 1:30PM I added some tiles to the control and chilled tanks.

Chilled treatment on the left and controls on the right

Chilled treatment on the left and controls on the right

It's cold!

Tiles added to treatments. The numbers represent oysters on that tile after sampling for gene expression. F = front of tile, S = side, B= back
Control
20 hours 0degC
3 hours 0degC
HCA5: 12F, 1S, 13B
HCA13: 9F, 1S
HCA11: 10F, 3S
HCB2: 7F, 2S, 8B
HCA12: 8F, 1S
SSA9: 4F, 1S (4 sampled)
HCA2: 1F, 8B
HCB5: 1
NFA6: 2F (4 sampled)
SSA4: 4F, 2S
HCB8: 2B
SSA13: 4F, 1R
SSA1: 14F, 2S, 1B
SSB11: 1F
SSA11: 0 (4 sampled)
SSA14: 4F, 1S
NFA4: 14F, 1B
SSB2: 0F, 2B
NFA11: 6F, 1B
NFA7: 3F, 1S
NFA9: 4F
NFA12: 3F, 1B
NFB2: 10F
NFA15: 11F, 1B
NFA14: 5F
NFA2: 26F, 1B
NFA?: 15F, 1B
Total HC: 52
Total SS: 19
Total NF: 63
Total HC: 22
Total SS: 17
Total NF: 41
Total HC: 13
Total SS: 5
Total NF: 2

At 4:00pm I sorted out 200 cultch set oysters from the chilled tank and added them to new silos in the control tank labelled with the population name and “3 hr chilled”. In addition to looking for differential mortality, we are interested in looking at temperature-induced gene expression. I put some oysters from each group in small (300 mL) cups with chilled water and then dissected 6 from each population and put whole body tissue in 1.5 mL tubes with RNALater. This took about 45 minutes total but I tried to alternate between populations while dissecting. I took pictures of most dissected oysters with a ruler. At 5:00pm I brought in one tile for each population from the chilled tank and dissected 4 oysters per population, giving 10 samples each. These samples were placed in the fridge overnight. The other ~150 oysters in the chilled tank were left there overnight to be sampled the next day.

Time
10:00AM
– Chiller turned on and set to 0degC.
– Ambient at 11degC
11:30AM
– Chilled side: 7degC
– 350 cultch set oysters placed in chilled water
12:15PM
– Chilled side: 4degC
1:00PM
– Chilled side: 0degC
1:30PM
-Added tiles to control and chilled tanks
4:00PM
-Sorted out ~200 per population; added to new silo in the control tank
– Dissected 10 samples per pop for gene expression