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== Expansion PCR (Quick test, round1) == * I did a quick test of expansion PCR on the three subset (20ul reaction). * I diluted 1st amplicon of previous probe set amplified by each primer set (by V4, V6, or eMIP_CA primers) into 2nM and will be as positive control for amplification. ** V4 set: I used MONOD GP1V4 as template. Mix 10ul of 20nM 1st round amplicon MONOD GP1V4 with 90ul H2O ** V6 set: I used MONOD V3 as template. Prepare 2nM template in the same way as V4 set. ** eMIP_CA: I used oligo from LC sciences used in Nature methods 2012. Mix 20ul of 10nM 1st round amplicon 4K LC Sciences with 80ul H2O 95C 30sec -> (95C 30sec -> 54C 45sec-> 72C 45sec) X 18-> 72C 3min -> 15C hold<br> [[File:2014-08-21_test_ExpPCR-90k_18x-r1.PNG|600px]] * Loaded 3ul of positive control and 5ul of amplicons from each subset in 6% TBE gel [[File:ZhangLab_2 2014-08-21 15hr 36min_90K-testExpPCR_r1.jpg|400px]] {| {{table}} class = wikitable | align="center" style="width:120px;background:#f0f0f0;"|'''Subset''' | align="center" style="width:100px;background:#f0f0f0;"|'''# oligos''' | align="center" style="width:100px;background:#f0f0f0;"|'''% in the pool''' |- | Set A||73,574||82.57 |- | Set B||12,994||14.58 |- | Set C||2,539||2.85 |} * Based on qPCR curve and gel image of the largest subset, subset A (82.57%), it looked like the amplification efficiency was not as high as previous batches of oligos from CustomArray. For optimization of expansion PCR in Blueprint project and other batches of oligos from CustomArray, qPCR curve started to shoot up ~6-7 cycles for 100nM oligo template and the band intensity was stronger by loading only 2.5ul of amplicons. * The smaller subset (set B and set C) had very faint intensity, so Rui wanted to know if increasing template amount would increase the intensity of the 150bp amplicons without inhibiting PCR reaction. I then tried a quick PCR again by increasing template concentration of set B to 150nM and reduce PCR cycle to 16 and 17 cycles. For subset C, I increase template concentration to 150nM and 200nM and ran PCR for 18 cycles since it takes only 2.85% of the pools. For subset A, I reduced PCR cycle number to 15 and 16 cycles, but used the same concentration of 100nM. ---- === Result of expansion PCR in Blueprint project (for comparison) === [[File:20130530_BSPPBlueprint_TESTExpensionPCR.png| 550px]] [[File:ZhangLab_2 2013-05-30 15hr 54min_TESTExpansionPCR.jpg| 200px]] [[File:20130530_BSPPBlueprint_ExpensionPCR.png| 550px]] ----
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