AlanFung:LabNotes/Sequencing/2009-11-22
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CVF-N
- Reamplify library and ship it out for shearing
A-22.5ng/ul B-12.8ng/ul C-6ng/ul D-5.4ng/ul
A 2ul B 1.76ul C 5.48ul D 32.85ul Total 15.32ul
- Amplify in 15 wells
' | ' | x15 | X1.1 | /2 | |
Library | 1 | - | - | 7.66 | |
2X Phusion | 50 | 750 | 825 | 412.5 | |
AmpF6.3NH2 (100uM) | 0.2 | 3 | 3.3 | 1.65 | |
AmpR6.3NH2 (100uM) | 0.2 | 3 | 3.3 | 1.65 | |
50X SYBR Green I | 0.2 | 3 | 3.3 | 1.65 | |
dH2O | 43 | 645 | 709.5 | 354.75 | |
- 98C 1min->5X(98C 10 sec, 58C 20 sec, 72C 20sec)->72C 5 min
Nanodrop-470ng/ul Concentration too high, probably due to other reagents in the solution Should be around 4.262ug for 5 cycles of amplification Purify with 3 qiaquick columns elute with 34ul EB buffer Nanodrop-