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== Expansion PCR (TEST) == {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''Final conc.''' | align="center" style="background:#f0f0f0;"|'''Volume for 3.5 rxn mix''' |- | 500nM seed oligoes||2.00 or 5.00||20nM or 50nM||0.00 |- | AP1V6 (100uM)||0.20||400nM||0.70 |- | AP2V6 (100uM)||0.20||400nM||0.70 |- | 2x KAPA SYBG fast MM||25.00||1x||87.50 |- | H2O||19.60||||68.60 |- | Total||45.00||||157.50 |} * Aliquot 45ul, add oligo template, and adjust total volume to 50ul with H2O * Add 2.5 ul of DMR330k Exp1, final conc. in expansion PCR is 1nM '''Program''' 95C 30sec -> (95C 30sec -> 54C 45sec-> 72C 45sec) X 15-> 72C 3min -> 15C hold [[File:20130530_BSPPBlueprint_TESTExpensionPCR.png| 550px]] * from qPCR curve, seemed like 20nM and 50nM didn't amplify very well comparing to PC as expected (similar to Matt's result using the oligos from the same source), so after PAGE verification, I will do expansion PCR with 100nM template. ''' PAGE analysis to verify amplified amplicons in 6% TBU gel ''' * Loaded 2.5ul of PCR products in TBU gel * I loaded amplified amplicons in TBU gel becuase I dodn't want to waste the TBU gel used for single strand seed oligos verification [[File:ZhangLab_2 2013-05-30 15hr 54min_TESTExpansionPCR.jpg| 200px]] Lane1: 10bp DNA ladder Lane2: O = 3ul of 500nM oligo before expansion PCR Lane3: PC (BSPP DMR330k Exp1) Lane4: 20nM BSPP _Blueprint after expansion PCR Lane5: 50nM BSPP _Blueprint after expansion PCR * From the gel image above, the expected band of ~150 bp was amplified. * The seed oligos from original tube before expansion PCR show a smear as observed in Matt' oligos. * There was the extra band in PC of DMR330k Exp1 >1kb. I have no idea where it come from. I opened this tube only twice, but every time I opened in the hood. I observed a faint band in the reaction containing BSPP Blueprint oligo with slightly larger size. * I continued to do expansion PCR by using oligo in the reaction 100nM, 2 tubes of 100ul reaction
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