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==== 2nd round PCR (QUICK TEST to monitor PCR cycle number) ==== * I did a quick test in small volume by using 2ul of bead purified 1st round PCR in total 10ul. Then split into 2X of 4.5ul and stopped based on qPCR curve. {| {{table}} border = 1 | align="center" style="width:140px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc''' | align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Final conc.''' | align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''12 rxn mix''' |- | Purified 1st round DNA||||||||||2.00||0.00 |- | 5X Phusion HF buffer||5||X||1||X||2.00||24.00 |- | dNTP mix||10||mM||0.25||mM||0.25||3.00 |- | TruS_F/R||10||uM||0.3||uM||0.30||3.60 |- | 50X SYBR||50||X||0.4||X||0.08||0.96 |- | Phusion HF||2||unit/ul||||||0.10||1.20 |- | H2O||||||||||5.27||63.24 |- | Total||||||||||10.00|| |} :- Aliquot 8 + 2ul DNA template :- Mix well. :- Split 4.5ul ::''98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 5, 6, and 7 cycles --> 72C for 2min'' '''KAPA Hyper prep''' :- Sample NC2 & NC3 stopped at 5X and 7X :- Sample 6P2 & 6P3 stopped at 5X and 6X '''NEXTflex''' :- Sample NC2 & NC3 stopped at 5X and 7X :- Sample 6P2 & 6P3 stopped at 5X and 7X :- Verify all PCR product in 6% TBE gel [[File:ZhangLab_2 2014-10-27 12hr 50min_2ndPCR-KAPA-NEXTflex-5x.jpg| 500px]] [[File:ZhangLab_2 2014-10-27 12hr 50min_2ndPCR-KAPA-NEXTflex-6x-7x.jpg| 500px]] * From the gel image, XXXXX :- I do 2nd round PCR in the large volume by using all 1st round PCR as template. I reduce template amount of 6P samples by KAPA Hyper Prep kit to 10% and increase 2nd round PCR to 6 cycles. For the rest samples, I use 20% of DNA template from 1st round PCR and ran 2nd round PCR for 8 cycles.
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