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===Amplification === * Perform amplification without Sybr. * Perform PCR with 12 cycles (fixed), 50 uL total volume. <nowiki> bis-cvt DNA 30.00 uL 10X Reaction buffer 5.00 uL dNTP, 10mM 1.25 uL TruSeq F/R, 10uM 1.50 uL PfuTurboCX 1.00 uL Nuclease free water 11.25 uL </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 2X Phusion HS Flex MM 5.00 uL TruSeq F/R, 10 uM 0.30 uL Nuclease free water 1.70 uL </nowiki> *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.
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