AlanFung:LabNotes/CTCF/2011-8-10
Jump to navigation
Jump to search
Objective[edit]
- Athurva's sequencing results came back, most of them failed and the one that worked reveals that the library is not bisulfite converted
- We will run a validation PCR to confirm the methylation status with the 3 set of primers CHR8,21 and 22
Protocol[edit]
- Prepare Master Mix for the 3 sets of primers
Content | Volume per reaction | MM |
ddH2O | 7 | 29.4 |
Primer F (3.3uM) | 6 | 25.2 |
Primer R (3.3uM) | 6 | 25.2 |
gDNA | 1 | Do not add to MM |
2X iQ SuperMix | 20 | 84 |
Total | 40 | 163.8 |
- Plate Layout
- Add 1ul of DNA to wells and add 39ul MM to each well accordingly
Chr8 | Chr21 | Chr22 |
Negative | Negative | Negative |
150 | 150 | 150 |
1500 | 1500 | 1500 |
Positive | Positive | Positive |
Perform PCR reaction in thermocycler
Step1 96C, 3m Step2 95C, 30s Step3 62C, 1m Step4 72C, 1m Step5 Go to step2 repeat 39 times Step6 72C, 5m Step7 4C, Forever
Results[edit]
File:ZhangLab 2 2011-08-10 17hr 01min.jpg
- It didn't look like the bisulfite conversion worked, no bands around 200bp for chr8 and 22 on sample 150 cells and 1500 cells, cannot draw conclusion from CHR21 for it has a lot of unspecific bands
- However Athurva has some promising results from sanger sequencing of his libraries, it appears that the sample was bisulfite converted.