PCR - Mac’s Bisulfite-Treated DNA

Per Mac’s request, ran a PCR on a set of bisulfite-treated DNA (in her gDNA 2014 box in small -20C):

• EV2.16 bisulfite

• EV2.20 bisulfite

• EV2.22 bisulfite

• EV2.24 bisulfite

• EV2.28 bisulfite

• EV2.29 bisulfite

• EV2.32 bisulfite

• EV2.33 bisulfite

DNA needed to be diluted. Diluted according to this sheet provided by Mac:

https://eagle.fish.washington.edu/bivalvia/070914bisulfite.pdf

NOTE: EV2.28 didn’t have sufficient DNA left to prepare the dilution according to Mac’s sheet. Instead, the remaining volume ofEv2.28 bisulfite DNA (0.5uL) was diluted in a total volume of 2.5uL to maintain the same dilution ratio.

Master mix calcs are here: 20140828 - PCR Mac Bisulfite Samples

Primers used were:

CgBS_733_26796Seq (SRID: 1598)

CgBS_733_26796R_5’biotin (SRID: 1596)

Cycling params:

1. 1. 95C - 10mins
2. 2. 94C - 30s
3. 3. 56C - 30s
4. 4. 72C - 30s
5. 5. Repeat steps 2 - 5 44 more times
6. 6. 72C - 10mins

Results:

Ladder used is O’GeneRuler 100bp DNA Ladder (ThermoFisher).

According to Mac, the expected band size is ~300bp. However, all samples are running at ~150bp. Mac is confused and does not know what to do.

UPDATE 20140902 - Realized I used the wrong forward primer! Will repeat PCR with correct primer. Wonder if Mac did the same thing…