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==== <u>2nd round PCR</u> ==== '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> :{| {{table}} border = 1 | align="center" style="width:180px;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;"|'''35 rxn mix''' |- | Purified 1st round DNA||||||||||20.00||0.00 |- | 5X Phusion HF buffer (CG)||5||X||1||X||10.00||350.00 |- | dNTP mix||10||mM||0.2||mM||1.00||35.00 |- | PCR_F||10||uM||0.3||uM||1.50||52.50 |- | PCR_R Index||5||uM||0.3||||3.00||0.00 |- | 50X SYBR||50||X||0.4||X||0.40||14.00 |- | Phusion HF||2||unit/ul||||||0.50||17.50 |- | H2O||||||||||13.60||476.00 |} :- Aliquot 27ul + 3ul of PCR_R Ind primer + 20ul template ::- I reduced amount of 1st PCR template in 2nd PCR ::''98C for 2min --> [98C for 10sec -> '''60C for 30sec''' -> 72C for 1min] X 6 cycles --> 72C for 2min''<br> ::- I stopped 2nd PCR very early as the qPCR curve getting saturated. :- I verified PCR product in 6% TBE gel [[File:ZhangLab_2 2014-12-20 16hr 07min_2ndPCR-6cycle-NP-gel1.jpg|400px]] [[File:ZhangLab_2 2014-12-20 16hr 07min_2ndPCR-6cycle-NP-gel2.jpg| 390px]] [[File:ZhangLab_2 2014-12-20 16hr 07min_2ndPCR-6cycle-NP-gel3.jpg|400px]] [[File:ZhangLab_2 2014-12-20 16hr 06min_2ndPCR-6cycle-YH-lower-PQ.jpg|480px]]
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