AlanFung:LabNotes/Sequencing/2009-11-24: Difference between revisions
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==Sequencing Library Construction with Single Adaptor Protocol-CVF-N Set== | ==Sequencing Library Construction with Single Adaptor Protocol-CVF-N Set== | ||
*To make 100ng of CVF-N set (A-D) | *To make 100ng of CVF-N set (A-D) | ||
* | *A-22.5ng/ul*1.5ul=33.75ng | ||
*B-12.8ng/ul*1.32ul=16.875ng | |||
*C-6ng/ul*4.11=24.64ng | |||
*D-5.4ng/ul*4.56=24.64ng | |||
*Total=100ng | |||
*To make 100ng of CVF-N set (A-B) | |||
*A-22.5ng/ul*2.96ul=66.6ng | |||
*B-12.8ng/ul*2.60ul=33.3ng | |||
*Total=100ng | |||
==PCR with dUTP and 6.3NH2 Primer== | ==PCR with dUTP and 6.3NH2 Primer== |
Revision as of 20:29, 24 November 2009
Sequencing Library Construction with Single Adaptor Protocol-CVF-N Set
- To make 100ng of CVF-N set (A-D)
- A-22.5ng/ul*1.5ul=33.75ng
- B-12.8ng/ul*1.32ul=16.875ng
- C-6ng/ul*4.11=24.64ng
- D-5.4ng/ul*4.56=24.64ng
- Total=100ng
- To make 100ng of CVF-N set (A-B)
- A-22.5ng/ul*2.96ul=66.6ng
- B-12.8ng/ul*2.60ul=33.3ng
- Total=100ng
PCR with dUTP and 6.3NH2 Primer
1 well | |
H2O | 46.2 |
Amp F6.3NH2(100uM) | 0.2 |
Amp R6.3NH2 (100uM) | 0.2 |
dUTP (1mM) | 2 |
50X SYBG I | 0.4 |
2X Master Mix | 50 |
Template | 1 |
Total | 100 |
- Qiaquick purification, elute with 32ul
USER Digestion
DNA | 30 |
USER | 3 |
Total | 33 |
S1 Nuclease Digestion
S1 Digestion | |
10X S1 Nuclease Buffer | 4 |
DNA after USER Digestion | 33 |
S1 Nuclease (10U/ul) | 1 |
ddH2O | 2 |
- 37C 10mins
- Minelute purification. Elute w/16uH2O