AlanFung:LabNotes/Sequencing/2009-11-22: Difference between revisions

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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;"|'''x4'''
| 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||32||645||709.5
| dH2O||43||645||709.5||354.75
|-
|-
|  
|  
|}
|}


*98C 1min->6X(98C 10 sec, 58C 20 sec, 72C 20sec)->72C 5 min
*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