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AlanFung:LabNotes/CTCF/2011-6-6
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==Perform bisulfite conversion on 200ng of gDNA== ==Protocol== ===Setup PCR Reaction=== *Add the components on ice in order while mixing gently. {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''amount per reaction''' |- | distilled water||34 |- | 10x pfuturbo cx reaction buffer||5 |- | dNTPs(10mM each DNTP)||1 |- | DNA template||2 |- | CHR8 F primer 3.3uM||3 |- | CHR8 R primer 3.3um||3 |- | sybr green 50x||1 |- | pfuturbo cx hotstart dna polymerase 2.5U/ul||1 |- | total reaction volume||50 |} *Add 39ul master mix to primers and templates **Perform Manufacturer protocol for PfuTurbo Cx Hotstart DNA Polymerase 95C 2m 95C 30s 52C 30s 72C 2m Plate Read Go to step 2 repeat for 29 times 72C 10m 15C hold ==Results== *Amplification failed again. ==CHR8 PCR amplification with iQ supermix== ==Objective== Confirm the bisulfite conversion works, iQ supermix has been the polymerase I used with the CHR primers and it works all the time. I will set up the reaction and confirm amplicons are successfully amplified before try with pfuturbo cx polymerase. ==protocol== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Amount per reaction''' |- | Distilled Water||18 |- | gDNA Template (bisulfite converted gDNA GM12878 done on 6/6/2011)||1 |- | CHR8 F primer 3.3uM||3 |- | CHR8 R primer 3.3um||3 |- | 2x iQ Super Mix||25 |- | Total reaction volume||50 |} 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 ==Results== [[File:060611_CHR8_iQ.jpg|500px]] *Amplification is successful. Will run a gel after I repeat the amplification with pfu cx polymerase
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