Repeated PCR from 20130919, but with a newly designed reverse primer. Primers were designed using MethPrimer (SR IDs: 1553, 1555). Primers were designed using MethPrimer:

Results:

No amplification of any samples BS-treated or non-treated.

Repeated PCR from 20130919, but with a newly designed set of primers that targets the desired region of C1q for sequencing (SR IDs: 1553, 1554). Primers were designed in Geneious.

Results:

No amplification of any samples BS-treated or non-treated.

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.