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==Continue on 8/29== *Talk to Dr. Zhang and he suggests that I can take 0.5ul of amplified samples and repeat the PCR, doing that will avoid over amplify all the samples. *Also me and Athurva need to come up with a plan on experiments to obtain a few aspects that we need to validate **whether bisulfite conversion is complete **whether the methylation level is accurately capture in the libraries **whether the resulting amplicons contain the right adaptors such that they can be sequenced on the illumina platform **whether all genomic regions are present at roughly equal abundance in the libraries **What can you get from the Sanger sequencing experiment, and what other experiments you need to do ===Library Amplification=== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''Volume''' | align="center" style="background:#f0f0f0;"|'''MM''' |- | Nuclease Free H2O||22.5||74.25 |- | 10uM Orange Primer||1||3.3 |- | 10uM Blue Primer||1||3.3 |- | Purified Amplicons||0.5||Do not add to MM |- | 2X Kapa SYBR Supermix||25||82.5 |- | Total||50||165 |} *Add 49.5ul MM to each reaction * Perform qPCR using the following reaction conditions: ** 95 C, 30 seconds (Denature) ** 35 cycles of: *** 95 C, 10 seconds (Denature) *** 58 C, 30 seconds (Anneal) *** 72 C, 3 minutes (Extension) ** 72 C, 5 minutes (final extension) ** Hold at 4 C ===Minelute=== *Minelute with 15ul EB buffer *Nanodrop Result **NTC:25.9ng/ul **150:94.1ng/ul **300:85.5ng/ul ===PAGE Gel Analysis=== [[File:ZhangLab_2 2011-08-29 18hr 51min.jpg|300px]]
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