AlanFung:LabNotes/Sequencing/2009-11-24: Difference between revisions
Jump to navigation
Jump to search
>Alan6017518 |
>Alan6017518 |
||
(5 intermediate revisions by the same user not shown) | |||
Line 1: | Line 1: | ||
==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== | ||
{| {{table}} | {| {{table}} | ||
| || | | A-D||A-D||X2||||A-B||X2 | ||
|- | |- | ||
| H2O||46. | | H2O||41.46||82.91||||41.64||83.28 | ||
|- | |- | ||
| Amp F6.3NH2(100uM)||0.2 | | Amp F6.3NH2(100uM)||0.2||0.4||||0.2||0.4 | ||
|- | |- | ||
| Amp R6.3NH2 (100uM)||0.2 | | Amp R6.3NH2 (100uM)||0.2||0.4||||0.2||0.4 | ||
|- | |- | ||
| dUTP (1mM)||2 | | dUTP (1mM)||2||4||||2||4 | ||
|- | |- | ||
| 50X SYBG I||0.4 | | 50X SYBG I||0.4||0.8||||0.4||0.8 | ||
|- | |- | ||
| 2X Master Mix||50 | | 2X Master Mix||50||100||||50||100 | ||
|- | |- | ||
| Template|| | | Template||5.75||11.49||||5.56||11.12 | ||
|- | |- | ||
| Total||100 | | 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 | *Qiaquick purification, elute with 32ul | ||
Line 38: | Line 50: | ||
| | | | ||
|} | |} | ||
*37C 2 hours | |||
==S1 Nuclease Digestion== | ==S1 Nuclease Digestion== | ||
{| {{table}} | {| {{table}} | ||
Line 55: | Line 69: | ||
*37C 10mins | *37C 10mins | ||
*Minelute purification. Elute w/16uH2O | *Minelute purification. Elute w/16uH2O | ||
Latest revision as of 21:51, 25 November 2009
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