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AlanFung:LabNotes/CTCF/2011-8-10
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==Objective== *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== *Prepare Master Mix for the 3 sets of primers {| {{table}} | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''Volume per reaction''' | align="center" style="background:#f0f0f0;"|'''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 {| {{table}} | align="center" style="background:#f0f0f0;"|'''Chr8''' | align="center" style="background:#f0f0f0;"|'''Chr21''' | align="center" style="background:#f0f0f0;"|'''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== [[File:ZhangLab_2 2011-08-10 17hr 01min.jpg|500px]] *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.
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