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==Continue on 9/1== *Randomly pick colonies and inoculate into 10ul of dH2O *Heat denature the colony suspension at 94C for 10min, quickly transfer to cold block {| {{table}} | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''Volume per Reaction''' | align="center" style="background:#f0f0f0;"|'''MM''' |- | Template||2||Do not add to MM |- | Nuclease Free Water||8.5||69.7 |- | M13 F (200ng)||1||8.2 |- | M13 R (200ng)||1||8.2 |- | Taq2X||12.5||102.5 |- | Total||25||188.6 |} *Aliquot 2ul of samples to a new 0.2ml tube *Add 23 ul MM to each sample *Vortex and spin down *Perform the following reaction in a thermo cycler **95C 2min **95C 30sec **60C 30sec **72C 1min **Go to step 2 for 29 cycles **72C 5min **Hold at 4C ===Page Gel Analysis=== [[File:ZhangLab_2 2011-09-01 14hr 58min.jpg|400px]] *It is abnormal to have streaking, it could be be to contamination from other colones, or just artifacts, but the sample amplicons (~600-800bp) are the dominant. Move on with purification and send out for sanger sequencing *Size of pCR Blunt vector is 270bp size of libraries are (175-800bp), so expected amplicons size are 445-1070bp, we are seeing amplicons between 600-800bp, it looks like we have the right size of amplicons {| {{table}} | align="center" style="background:#f0f0f0;"|'''Label''' | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''ng/ul''' | align="center" style="background:#f0f0f0;"|'''20ng''' | align="center" style="background:#f0f0f0;"|'''dh2o''' | align="center" style="background:#f0f0f0;"|'''5uM M13R''' |- | HL01||150-1||7.2||2.78||7.22||5 |- | HL02||150-2||7||2.86||7.14||5 |- | HL03||150-3||8.5||2.35||7.65||5 |- | HL04||150-4||9.8||2.04||7.96||5 |- | HL05||300-1||9.5||2.11||7.89||5 |- | HL06||300-2||5.9||3.39||6.61||5 |- | HL07||300-3||9.3||2.15||7.85||5 |- | HL08||300-4||6.7||2.99||7.01||5 |}
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