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===Amplification === * Perform amplification with and without Sybr. I reduced reaction volumes by half each. * Perform PCR with 12 cycles (fixed) <nowiki> bis-cvt DNA 15.000 uL 10X Reaction buffer 2.500 uL dNTP, 10mM 0.625 uL TruSeq F/R, 10uM 0.750 uL 50X Sybr 0.400 uL (0 for no sybr) PfuTurboCX 0.500 uL Nuclease free water 5.225 uL (5.625 for no sybr) </nowiki> *Mix well *Program: 95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 12 cycles --> 72C for 2min *Purify with 1X AMPure beads, incubate DNA with beads 15 minutes, wash twice with freshly prepared 80% ethanol, dry beads for 20 minutes (?)) *Elute with 20 uL EB buffer *Continue to 2nd round PCR. No PAGE Verification * Test 2nd round PCR * Noi's protocol: <nowiki> Purified 1st round DNA 3.00 uL 5X Phusion HF buffer 2.00 uL dNTP mix, 10mM 0.25 uL TruSeq F/R, 10 uM 0.30 uL 50X Sybr 0.08 uL (0 for no sybr) Phusion HF 0.10 uL Nuclease free water 4.27 uL (4.35 for no sybr) </nowiki> * I couldn't find Phusion HF, so I used Phusion Hotstart Flex Master Mix instead. *Mix well *Program: 98C for 2min β [98C for 10sec β 60C for 30sec β 72C for 1min] X 9X cycles β 72C for 2min *Load everything to 6% gel and run at 225 V for 30 min. * Once PCR cycle is determined (8-9 cycles?), prepare PCR for all the samples and then purify with AMPure beads. Run gel to also determine the pooling ratios and perform size selection for 150-400 bp bands. * Gel images: ** (+) is with sybr added, (-) is without sybr added. The amplification patterns are similar on gel, except in some cases sybr added more intensity. ** I remember that PfuTurboCx have less amplification with sybr, its not possible that sybr would increase the amplification efficiency, so I think the sybr green may have helped to make the fluorescence brighter for the DNA amplified with sybr. For now, I don't see evidencee that the sybr have an effect on amplification, so I could amplify with or without sybr. [[File:Dinh_1ng_RRBS_09182015_gel1.jpg | 300px]] [[File:Dinh_1ng_RRBS_09182015_gel2.jpg | 300px]]
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