20140828

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:

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

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


Results:

external image 20140829%20-%20Gel%20EV2%20bisulfite%20PCR.png

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.