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==== <u>1st round PCR</u> ==== '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> * I used all eluted bisDNA in the 1st round PCR :{| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc''' | align="center" style="width:80px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Final conc.''' | align="center" style="width:80px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''50 rxn mix''' |- | Bis-cvt DNA||||||||||30.00||0.00 |- | 10X Reaction buffer||10||X||1||X||5.00||250.00 |- | dNTP mix||10||mM||0.3||mM||1.50||75.00 |- | TruF/R||10||uM||0.3||uM||1.50||75.00 |- | PfuTurbo Cx||2.5||Unit/ul||1||unit||0.40||20.00 |- | 50X SYBG||50||X||0.4||X||0.40||20.00 |- | H2O||||||||||11.20||560.00 |- | Total||||||||||50.00|| |} :- Aliquot 20ul, add 30ul of bisulfite-treated adaptor-ligated DNA ::''95C for 2min --> [95C for 20sec -> '''60C for 30sec''' -> 72C for 1min] X 24 cycles --> 72C for 2min''<br> * I stopped 1st round PCR at 17 cycles as the qPCR curve was close to saturate. * I was still worried about the cycle number as 17 cycles was much less than 25 cycles by Tang's group. However, I could not let it over amplified of adaptor dimers as well. ::- I took 5ul of 1st PCR product of all samples for PAGE verification. * This might take a lot of time and TBE gel to verify. However, I want to observe the result in every single step to be able to interpret the result and get optimum condition for scRRBS.
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