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.

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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.

 

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.

Monday 9/28/15

Do AlfI digestion of ethanol precipations from last week.
  1. SS2_3
  2. HC1_1
  3. NF1_1
  4. NF1_2
  5. SS2_6
  6. SS2_7
  7. SS2_8
  8. HC1_5
  9. HC1_6
  10. HC1_7
  11. HC1_8
  12. HC1_9
  13. HC1_10
  14. NF1_4
  15. NF1_5
  16. NF1_6
  17. NF1_7
  18. NF1_8
1x
20x
10x buffer R
1.2 uL
24
150 uM SAM
.8
16
AlfI (2 U/uL)
.5
10
water
1.5
30

Did ligation of same samples. Left for 2.5 hours at 16degC and did 10 minutes at 65degC following Matz protocol.

  • 1st make new adaptors.
  • Adaptor 1
    11x
    20x
    5ILL-NR
    .6 uL
    1.1
    Anti-ILL
    .6 uL
    1.1
    NFW
    58.8 uL
    106.91
    Adaptor 2
    11x
    3ILL -NR
    .6 uL
    Anti-ILL
    .6 uL
    NFW
    58.8 uL
    Master mix
    1x
    20
    2 uM Adaptor 1
    5 uL
    100
    2 uM Adaptor 2
    5 uL
    100
    T4 ligase
    1 uL
    20
    T4 ligase buffer with 10 mM ATP
    4 uL
    80
    NFW
    25
    500

    40 uL master mix + 10 uL digestion product

Friday 9/18/15

Ligation of adaptors to digested samples.

Need to make new adaptors each time. Recipe below is for 10 samples (+1 for pipet error) to make adaptors at 2 uM.  Combine and let sit for 10 minutes at room temp – I made sure stocks were completely melted.

Adaptor 1
11x
100 um 5ILL-NR
.6 uL
100 um Anti-ILL
.6 uL
NFW
58.8 uL
Adaptor 2
11x
100um 3ILL -NR
.6 uL
100um Anti-ILL
.6 uL
NFW
58.8 uL
Ligation reaction master mix
1x
11x
2 uM Adaptor 1
5 uL
55
1 uM Adaptor 2
5 uL
55
T4 ligase
1 uL
11
T4 ligase buffer with 10 mM ATP
4 uL
44
NFW
25
275

*Note: the Meyer protocol calls for 1.0 uL rATP  and 4.0 uL T4 ligase buffer. My buffer had 10 mM rATP included, so I just used 25 uL of NFW instead of 24uL and did not add more rATP.

40 uL master mix with 10 uL of digested DNA. Held at 16degC for 2 hours and 15 minutes. Put in -20C.