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==Prepare SuperMix== *Prepare 3 tubes for 3 different primers mix {| {{table}} | ||CHR22||CHR21||Chr8 |- | 2X iQ Super Mix||44||44||44 |- | Primer F (3.3uM)||13.2||13.2||13.2 |- | Primer R (3.3uM)||13.2||13.2||13.2 |- | |} *Aliquot 32ul to each well +8ul sample =40ul Total Perform PCR reaction in real-time thermocycler {| {{table}} | Setup |- | CV-Fibr (CHR22) |- | CV-Fibr (CHR21) |- | CV-Fibr (CHR8) |- | Jurkat (CHR22) |- | Jurkat (CHR21) |- | Jurkat (CHR8) |- | |} 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 8ul h2o+1.5ul 6x loading dye+0.5ul 25bp ladder 6.5ul h2o+1.5ul 6X loading dye+2ul sample Run at 250V for 20min {| {{table}} | Lane||Setup|| |- | 1||LM Ladder|| |- | 2||CV-Fibr (CHR22)|| |- | 3||CV-Fibr (CHR21)|| |- | 4||CV-Fibr (CHR8)|| |- | 5||Jurkat (CHR22)|| |- | 6||Jurkat (CHR21)|| |- | 7||Jurkat (CHR8)|| |- | 8||NA12878||PCR_AMP F/R 6.3r Sol |- | 9||NA18507||PCR_AMP F/R 6.3r Sol |- | 10||NA20431||PCR_AMP F/R 6.3r Sol |- | 11||Jurkat_New||PCR_AMP F/R 6.3r Sol |- | 12||Jurkat _Old||PCR_AMP F/R 6.3r Sol |- | |} [[Image:ZhangLab_2 2010-01-07 14hr 55min.jpg]] *CT Conversion Works *PCR with sequencing adapters works on NA20431, mix NA20431 (6.3 & 6.3r Sol) and perform size selection
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