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.

Advertisements

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.

10/23/15

Common Garden Project

DNA extraction from Olympia oyster broodstock

  1. NF2_1
  2. NF2_2
  3. NF2_3
  4. NF2_4
  5. NF2_5
  6. NF2_6
  7. NF2_7
  8. NF2_9
  9. NF2_10
  10. NF2_11
  11. NF2_12
  12. NF2_13
  13. NF2_14
  14. NF2_15
  15. NF2_16
  16. NF2_17
  17. NF2_18
  18. NF2_19
  19. NF2_20
  20. NF3_1
  21. NF3_2
  22. NF3_3
  23. NF3_4
  24. NF3_5

Did not find NF2_8…

Olympia Oyster Population Structure Project

I started preparing genotype-by-sequencing libraries using the dried down samples from 10/21/15. Did an ApeKI digestion and ligation of adaptors.

Digestion:

1x 50x
NEB Buffer 3 2.μL 100
ApeKI 1 μL 50
H2O 17 μL 850

Added 20 uL of digestion master mix to each of the 48 wells. Incubated for 2 hours at 75degC then held at 4degC until ligation master mix was ready.

1x 51x
T4 Ligase Buffer 5 μL 255
T4 Ligase 1.6 μL 81.6 μL
H2O 23.4 μL

Added 30 uL of ligation mastermix to each well to give 50 uL total. Incubated at 19degC for 1 hour and 65degC for 20 minutes. Cooled to 0degC and put in freezer.