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='''EZ DNA Methylation Direct-GM20431 PCR analysis '''= ==Objective== *Confirmation of the bisulfite conversion of GM20431 for 1,000 cells *Continuation of lowering cell number *Increase low mass ladder volume to 2uL for a better reference *PCR A B C D E ----------------------------- Taq 2X Master Mix 20 20 20 20 20 Primer F (3.3uM) 6 6 6 2 2 Primer R (3.3uM) 6 6 6 0 0 gDNA 0.5 0.5 0.5 0.5 0 RNAse free H20 7.5 7.5 7.5 15.5 16 ----------------------------- 40uL A-CHR22 F/R B-CHR21 F/R C-CHR8 F/R D-H18S F/R E-H18S F/R 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 *Gel Electrophoresis Well 1 2 3 4 5 6 7 8 ------------------------------------------------------------- Content Blank CHR22 CHR21 CHR8 H18S H18S Ladder Sample 0 6 6 6 6 6 2 0 0.5X TBE Buffer 0 4 4 4 4 4 3 0 6X Loading Dye 0 4 4 4 4 4 3 0 ------------------------------------------------------------- **Run at 135 V for 20 min. ==Results== [[Image:ZhangLab_2 2009-03-10 11hr 49min_crop.jpg]] *Clear bands showed up around 200bp for primer CHR22 only *Positive control showed dimmer band around than previous PCR *Negative control still showing up and its brighter than usual, it is even brighter than the +ve control *Ladder is showing up better but still not clear enough ==Troubleshoot== *Run a side by side bisulfite conversion of concentration of gDNA and *Samples <2000 cells should be treated with less M-Digestion Buffer according to the Zymo protocol *Increase the ladder volume to 3uL *Increase the concentration of the agarose gel to 1.5%-2%
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