Tag Archives: bisulfite PCR

PCR – Mac’s Bisulfite-Treated DNA

Realized that the PCR performed on 20140828 used the incorrect forward primer! As such, am repeating as before, but with the correct forward primer:

CgBS_733_26796F (SRID: 1597)

NOTE: Nothing left of sample EV2.28 bisulfite, so this was not run.

Results:

Ladder – O’GeneRuler 100bp Ladder (ThermoFisher)

Samples are loaded in numerical order from left to right, with a NTC sample before the second ladder.

All samples ran at ~275bp, which is larger than the previous gels. Confirmed with Mac that this gel looks correct. Will contact Cassie at Fred Hutchinson to go forward with PyroMark sequencing of these products.

Evidently, it would seem that Mac (and I) used the incorrect primer set when performing this PCR most recently. Doh!

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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

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…

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PCR – Lake Trout C1q

Ran PCR on lake trout DNA and lake trout bisulfite-converted DNA. Used primers SRIDs: 1551 and 1552. DNA was isolated by Caroline Storer on 4/4/2011 and bisulfite converted on 4/7/2011. Master mix and cycling params are here:

http://eagle.fish.washington.edu/Arabidopsis/Notebook%20Workup%20Files/20130919-01.jpg

Samples:

Lake Trout Lean_6 Liver

Lake Trout Lean_7 Liver

Lake Trout Siscowet_6 Liver

Lake Trout Siscowet_7 Liver

Bisulfite converted DNA from the four samples listed above.

Results:

Lane 1: Hyperladder II (Bioline)

Lane 2: Lean6

Lane 3: Lean6 BS

Lane 4: Lean7

Lane 5: Lean7 BS

Lane 6: Siscowet6

Lane 7: Siscowet6 BS

Lane 8: Siscowet7

Lane 9: Siscowet7 BS

All the non-BS converted samples amplified as expected, producing a band of ~560bp. However, none of the BS-converted DNA produced any amplification. It is likely an issue with the primer sequences and the resulting conversion of the gDNA.

Will look at Caroline Storer’s notebook entries for her work on this and try to evaluate what has already been done.

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