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== Amplification == * I normally do a quick test PCR in small volume 20ul with 10% v/v of captured DNA. This to monitor PCR cycle number and check if we get the right amplified product by running PCR product in 6% TBE gel without bead/colum purification. If the experiment has been optimized for a certain probe set, may not need to do quick qPCR check. This time I want to compare between the two enzymes, Phusion Flash High-Fidelity MM and KAPA HiFi Hotstart ReadyMix. ==== Quick PCR ==== * I randomly picked few samples from normal controls and cancer patient samples for a quick test PCR, including NC-1, NC-2, NC-3 NC-4, PCP-2, PCP-3, PCP-4, PCP-5, ^T1 from previous exp. as positive control and NTC. {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Stock conc.''' | align="center" style="width:80px;background:#f0f0f0;"|'''Final conc. In PCR''' | align="center" style="width:80px;background:#f0f0f0;"|'''1x rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''13x rxn''' |- | Captured template||100%||10%||2.00||0.00 |- | 10uM AmpF6.4Sol||10uM||0.2uM||0.40||5.20 |- | 10uM AmpR6.3.IndX 8||10uM||0.2uM||0.40||5.20 |- | 2X Phusion Flash Hi-Fidelity MM or KAPA HiFi Hotstart ReadyMix ||2X||1X||10.00||130.00 |- | 50X SYBR||50X||0.4X||0.16||2.08 |- | H2O||||||7.04||91.52 |- | Total||||||20.00|| |} :- Aliquot 18ul + 2ul of captured DNA template * Note: In PCR, I included one of positive control (PTC) of captured templates from gDNA of tumor tissue sample to compare the size of amplified amplicons. ::- ''98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x10-15 cycles -> 72C 3min'' * Note that I just realized that KAPA HiFi Hotstart ReadyMix requires 2-3min for hot start activation. I paused the machine for 1 min a9 98C during denaturing step. I was not sure if this will result in not-fully active DNA polymerase for KAPA. :- In this experiment, we stopped at 19 cycles :- Loaded 5ul of PCR product in 6% TBE gel without purification (Run at 250 volts in 0.5X TBE buffer for 25min) [[File:ZhangLab_2 2014-12-21 17hr 59min_PhusionFlash-quickPCR-LMS-WGBS-5ul.jpg| 450px]] [[File:ZhangLab_2 2014-12-21 17hr 59min_KAPAHiFi-quickPCR-LMS-WGBS-5ul.jpg| 450px]] * The pattern of PCR products was consistent between the two enzymes. However, Phusion Flash Hi-Fidelity showed stronger intensity, so I will keep using this enzyme for the future. This should be note that a lower signal from KAPA HiFi Hotstart ReadyMix might be due to too short denaturation. However, I had no time to repeat experiment. * I continued to amplified the rest of captured DNA in large volume (200ul --> split 2X of 100ul reaction) for 20 cycles using Phusion Flash Hi-Fidelity. * I pooled 80ul from each well (total 160ul) and purified with 0.7X AMPure bead. * Resuspended with 60ul H2O and quantified by PAGE quantification. [[File:ZhangLab_2 2014-12-28 04hr 25min_PQ-gel1.jpg|450px]] [[File:ZhangLab_2 2014-12-28 04hr 25min_PQ-gel2.jpg|450px]] [[File:ZhangLab_2 2014-12-28 04hr 25min_PQ-gel3.jpg|450px]] [[File:ZhangLab_2 2014-12-28 04hr 25min_PQ-gel4.jpg|475px]] [[File:ZhangLab_2 2014-12-28 04hr 25min_PQ-gel5.jpg|450px]] [[File:ZhangLab_2 2014-12-28 04hr 25min_PQ-gel6.jpg|450px]]
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