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== Expansion PCR == {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''Final conc.''' |- | 20nM LC Sciences Oligoes||10.00||1nM |- | eMIP_CA1_F (100uM)||0.80||400nM |- | eMIP_CA1_R (100uM)||0.80||400nM |- | 2x Kapa SYBG MM||100.00||1x |- | H2O||88.40|| |- | Total||200.00|| |} '''Program'''<br> 95C 30sec -> (95C 5sec -> 52C 60sec-> 72C 30sec) x '''5''' -> (95C 5sec -> 60C 30sec-> 72C 30sec) x '''10''' -> 72C 2min -> 15C hold * Primer info. ** eMIP_CA1_F: TGCCT'''AGGACCGGATCAACT''' (TGCCT overhang), Tm of the short one = 53.06C, the long one = 63.28C ** eMIP_CA1_R: GAGCT'''TCGGTTCACGCAATG''' (GAGCT overhang), Tm of the short one = 54.41C, the long one = 62.95C * Note: ** The first five cycles, Tm of each primer was calculated without 5 nt overhang, then the Tm was increased based on the Tm of the full length primer ** The number of cycles will be monitored [[File:Expansion-PCR_2011_10_07.png| 500px]] * Purified with Qiaquick column (2 columns) and elute with EB buffer volume 50ul each --> total volume = 100ul * Measure DNA conc. with Nanodrop: 18ng/ul or 295.5nM * Dilute 1st round amplicon to 10nM volume 1000ul (mix 33.89ul of 295.5nM 1st round amplicon with 966.11ul H2O)--> for using as the template for the future amplification * Perform production PCR
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