AlanFung:LabNotes/Sequencing/2009-11-22: Difference between revisions
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>Alan6017518 (→CVF-N) |
>Alan6017518 (→CVF-N) |
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==CVF-N== | ==CVF-N== | ||
*Reamplify library and ship it out for shearing | *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 | |||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''''' | | align="center" style="background:#f0f0f0;"|'''''' | ||
| align="center" style="background:#f0f0f0;"|'''''' | | align="center" style="background:#f0f0f0;"|'''''' | ||
| align="center" style="background:#f0f0f0;"|''' | | align="center" style="background:#f0f0f0;"|'''x15''' | ||
| align="center" style="background:#f0f0f0;"|'''X1.1''' | | align="center" style="background:#f0f0f0;"|'''X1.1''' | ||
| align="center" style="background:#f0f0f0;"|'''/2''' | |||
|- | |- | ||
| Library||1||-||-|| | | Library||1||-||-||7.66|| | ||
|- | |- | ||
| 2X Phusion||50||750||825 | | 2X Phusion||50||750||825||412.5 | ||
|- | |- | ||
| AmpF6.3NH2 (100uM)||2||3||3.3 | | AmpF6.3NH2 (100uM)||0.2||3||3.3||1.65 | ||
|- | |- | ||
| AmpR6.3NH2 (100uM)||2||3||3.3 | | AmpR6.3NH2 (100uM)||0.2||3||3.3||1.65 | ||
|- | |- | ||
| 50X SYBR Green I||2||3||3.3 | | 50X SYBR Green I||0.2||3||3.3||1.65 | ||
|- | |- | ||
| dH2O|| | | dH2O||43||645||709.5||354.75 | ||
|- | |- | ||
| | | | ||
|} | |} | ||
*98C 1min-> | *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 (Start off w/ 133.2ng) | |||
Purify with 3 qiaquick columns elute with 34ul EB buffer | |||
Nanodrop-29ng/ul * 30ul =870ng |
Latest revision as of 07:22, 23 November 2009
CVF-N[edit]
- 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 (Start off w/ 133.2ng) Purify with 3 qiaquick columns elute with 34ul EB buffer Nanodrop-29ng/ul * 30ul =870ng