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== Production PCR == {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1 rxn''' | align="center" style="background:#f0f0f0;"|'''25x rxn mix''' |- | 1st round amplicon (10nM)||0.20||5.00 |- | eMIP_CA1_F (100uM)||0.40||10.00 |- | eMIP_CA1_R (100uM)||0.40||10.00 |- | 2x KAPA SYBG fast MM||50.00||1,250.00 |- | H2O||49.00||1,225.00 |- | Total||100.00||2,500.00 |} * Aliquot 100ul to 3 8-striptubes '''Program'''<br> 95C 30sec -> (95C 5sec -> 60C 30sec-> 72C 30sec) x 14 -> 72C 2min -> 15C hold<br> [[File:2013_03_08_ProductionPCR_12koligos.png| 550px]] * Note: ** I increased extension time to 30sec) ** Number of cycle will be monitored * Perform EtOH precipitation of the 24 rxn PCR in 3x 15mL tube * 800ul amplified amplicons + 80ul 3M NaOAc pH5.5 + 2.6ul Glycoblue and 2mL 100% EtOH * Incubate at -80C for 30min --> centrifuge at 3,000 rpm for 30 min, wash with 75% EtOH * Resuspend with 100ul each tube * Re-purify with Qiaquick column (3 columns) and elute with 50ul EB buffer each --> total volume 150ul purified amplicons * Measure conc. with Nanodrop --> 89.6ng/ul, yield ~13.44ug.
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