AlanFung:LabNotes/Sequencing/2009-11-24
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Sequencing Library Construction with Single Adaptor Protocol-CVF-N Set[edit]
- 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[edit]
A-D | A-D | X2 | A-B | X2 | |
H2O | 41.46 | 82.91 | 41.64 | 83.28 | |
Amp F6.3NH2(100uM) | 0.2 | 0.4 | 0.2 | 0.4 | |
Amp R6.3NH2 (100uM) | 0.2 | 0.4 | 0.2 | 0.4 | |
dUTP (1mM) | 2 | 4 | 2 | 4 | |
50X SYBG I | 0.4 | 0.8 | 0.4 | 0.8 | |
2X Master Mix | 50 | 100 | 50 | 100 | |
Template | 5.75 | 11.49 | 5.56 | 11.12 | |
Total | 100 | 200 | 100 | 200 | |
PCR program: 94 °C for 3 min, 5 cycles of 94 °C for 45 sec, 55 °C for 45 sec, 72 °C for 45 sec, and finally 72 °C for 3 min.
- Qiaquick purification, elute with 32ul
USER Digestion[edit]
DNA | 30 |
USER | 3 |
Total | 33 |
- 37C 2 hours
S1 Nuclease Digestion[edit]
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