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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