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== PCR Amplification == * I performed PCR on half of captured DNA (10ul) in total 50ul PCR reaction (20% v/v in PCR) {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''26 rxn mix''' |- | Captured template||10.00||0.00 |- | 10uM AmpF6.4Sol||1.00||26.00 |- | 10uM AmpR6.3.IndX (X=1-24)||1.00||0.00 |- | 2X KAPA SYBR MM||25.00||650.00 |- | H2O||13.00||338.00 |- | Total||50.00|| |} * Aliquot 39ul + 1ul AmpR6.3 IndX + 10ul DNA template {| {{table}} class =wikitable | align="center" style="background:#f0f0f0;"|'''Samples''' | align="center" style="width:50px; background:#f0f0f0;"|'''C1''' | align="center" style="width:50px; background:#f0f0f0;"|'''C2''' | align="center" style="width:50px; background:#f0f0f0;"|'''A1''' | align="center" style="width:50px; background:#f0f0f0;"|'''A2''' | align="center" style="width:50px; background:#f0f0f0;"|'''NTC''' | align="center" style="width:50px; background:#f0f0f0;"|'''C1 no GP1V4''' | align="center" style="width:50px; background:#f0f0f0;"|'''A1 no GP1V4''' | align="center" style="width:50px; background:#f0f0f0;"|'''A2 no GP1V4''' |- | Strip 1: Stoffel||Ind_1||Ind_2||Ind_3||Ind_4||Ind_5||Ind_6||Ind_7||Ind_8 |- | Strip 2: Hemo KlenTaq||Ind_9||Ind_10||Ind_11||Ind_12||Ind_13||Ind_14||Ind_15||Ind_16 |- | Strip 3: ILMN's Pol||Ind_17||Ind_18||Ind_19||Ind_20||Ind_21||Ind_22||Ind_23||Ind_24 |} '''Program (Eppendorf Realplex)''' 98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min * I stopped all samples at 21x cycles except sample C1,C2 with Hemo KlenTaq/ Illumina' Polymerase at 22x cycles. * Verify 3ul of PCR product in 6% TBE gel (no AMPure bead purified) '''Stoffel Fragment'''<br> [[File:ZhangLab_2 2014-05-26 13hr 01min_GP1v4_C1C2A1A2NTC_Stoffel.jpg| 400px]]<br> '''Hemo KlenTaq'''<br> [[File:ZhangLab_2 2014-05-26 13hr 01min_GP1v4_C1C2A1A2ntc_HemoKlenTaq.jpg| 400px]]<br> '''Illumina's Polymerase'''<br> [[File:ZhangLab_2 2014-05-26 13hr 01min_GP1v4_C1C2A1A2ntc_ILPol.jpg| 400px]]<br> * ** = over-amplified * From the result above, it's clear that cancer samples with '''Stoffel fragment''' and '''Hemo Klentaq''' showed a smear between 350~390bp. I think there is the variation of DNA input in different tubes because there is more background in the samples with strong signal. * Amplified DNA from Illumina showed the same pattern in three enzyme, but has highest background in Hemo KlenTaq * With Illumina's polymerase, cancer samples showed discrete bands of ~350, 390 and above 400bp similar to amplified DNA from Illumina. ---- * Example of GP1V4 capture on cancer and blood samples I did experiment on 2013-12-24 ('''after AMPure bead purified''') [[File:ZhangLab_2 2013-12-25 19hr 44min_GP1_V4_1-8_PQ.jpg| 400px]]
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