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== Expansion PCR in a large volume == * I do expansion PCR of the 3 subset in total volume 300ul for each subset. I use 100nM template for Subset A and B and run for 17 cycles. For subset C, I use 200nM template and run for 18 cycles <u>'''Set A (V6 primers)'''</u> {| {{table}} border = 1 | align="center" style="width:180px;background:#f0f0f0;"|'''Components''' | align="center" style="width:100px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:100px;background:#f0f0f0;"|'''Final conc.''' |- | 3,142.7nM seed oligoes||9.55||100nM |- | AP1V61U (100uM)||1.20||400nM |- | AP2V6 (100uM)||1.20||400nM |- | 2x KAPA SYBG fast MM||150.00||1x |- | H2O||138.05|| |- | Total||300.00|| |}<br> <u>'''Set B (V4 primers)'''</u> {| {{table}} border = 1 | align="center" style="width:180px;background:#f0f0f0;"|'''Components''' | align="center" style="width:100px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:100px;background:#f0f0f0;"|'''Final conc.''' |- | 3,142.7nM seed oligoes||9.55||100nM |- | AP1V41U (100uM)||1.20||400nM |- | AP2V4 (100uM)||1.20||400nM |- | 2x KAPA SYBG fast MM||150.00||1x |- | H2O||138.05|| |- | Total||300.00|| |}<br> <u>'''Set C (eMIP_CA1 primers)'''</u> {| {{table}} border =1 | align="center" style="width:180px;background:#f0f0f0;"|'''Components''' | align="center" style="width:100px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:100px;background:#f0f0f0;"|'''Final conc.''' |- | 3,142.7nM seed oligoes||19.09||200nM |- | eMIP_CA1_F (100uM)||1.20||400nM |- | eMIP_CA1_R (100uM)||1.20||400nM |- | 2x KAPA SYBG fast MM||150.00||1x |- | H2O||128.51|| |- | Total||300.00|| |} * For each subset, I aliquot 50ul to 6X in strip tube 95C 30sec -> (95C 30sec -> 54C 45sec-> 72C 45sec) X 18-> 72C 3min -> 15C hold [[File:2014-08-22_ExpPCR_setA-B_17x.PNG| 500px]] [[File:2014-08-22_ExpPCR_setC_18x.PNG| 500px]] * Purified with Qiaquick column (3 columns for each) and elute with EB buffer volume 52ul each --> total volume left = ~140ul * Measure DNA conc. with Nanodrop for each subset * M.W. of dsDNA 148bp = 148bp * 650Da/bp = 96,200 g/mole {| {{table}} class = wikitable | align="center" style="width:180px;background:#f0f0f0;"|'''Subset''' | align="center" style="width:100px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:100px;background:#f0f0f0;"|'''260/230''' | align="center" style="width:100px;background:#f0f0f0;"|'''260/280''' | align="center" style="width:100px;background:#f0f0f0;"|'''Conc. (nM)''' |- | LMS cluster (set A)||10.83||5.75||2.49||110.87 |- | Selector LMS (set B)||9.46||3.75||2.15||96.85 |- | Selector chr21 (Set C)||13.89||4.17||2.05||142.20 |} * I will give set C to Rui for production PCR * Dilute 1st round amplicons of subset A and subset B to 20nM for using as the template for future production PCR<br> {| {{table}} border = 1 | align="center" style="width:180px;background:#f0f0f0;"|'''Subset''' | align="center" style="width:80px;background:#f0f0f0;"|'''Original conc. (nM)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Add EB buffer to 20nM''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total volume (ul)''' |- | LMS cluster (set A)||110.87||140||636.09||776.09 |- | Selector LMS (set B)||96.85||140||537.95||677.95 |} * Continued to production PCR <br>
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