Noi/NOTES/2011-9-1
Jump to navigation
Jump to search
Randomly tagging primer test
- Received 4 new primers for the project
- AmpF7NU.Sol
- AmpF7AU.Sol
- Syb_FP5A
- Syb_RP7
Tests:
- Does KAPA work?
- Does new F primer work for both Phusion and KAPA?
- Can we reduce primer conc. From 0.2uM to 0.02uM?
PCR set up
- Phusion, 0.2uM primer, 3 tubes of reactions : 1. old F primer (AmpF6.4.Sol), 2. new F primer (AmpF7AU.Sol), 3. NTC
- Note: NTC used old F primer (AmpF6.4.Sol)
Components | 1 rxn | 3.1x rxn mix P |
Circular DNA | 2.00 | 0.00 |
10uM AmpF6.4Sol or new F | 1.00 | 0.00 |
10uM AmpR6.3Sol | 1.00 | 3.10 |
2x Phusion | 25.00 | 77.50 |
50x SYBG | 0.50 | 1.55 |
H2O | 20.50 | 63.55 |
Total volume (ul) | 50 | 145.70 |
- aliquot 47ul, add 1ul of 10 uM F primer and 2ul of circular DNA
- KAPA, 0.2uM primer, 3 tubes of reactions: 1. old F primer (AmpF6.4.Sol), 2. new F primer (AmpF7AU.Sol), 3. NTC
- Note: NTC used old F primer (AmpF6.4.Sol)
Components | 1 rxn | 3.1x rxn mix K |
Circular DNA | 2.00 | 0.00 |
10uM AmpF6.4Sol or new F | 1.00 | 0.00 |
10uM AmpR6.3Sol | 1.00 | 3.10 |
2x KAPA | 25.00 | 77.50 |
H2O | 21.00 | 65.10 |
Total volume (ul) | 50 | 145.70 |
- aliquot 47ul, add 1ul of 10uM F primer and 2ul of circular DNA
- Phusion, 0.02uM primer 1 tube of reaction: old F primer
Components | ==1 rxn== |
Circular DNA | 2.00 |
1uM AmpF6.4Sol | 1.00 |
1uM AmpR6.3Sol | 1.00 |
2x Phusion | 25.00 |
50x SYBG | 0.50 |
H2O | 20.50 |
Total volume (ul) | 50.00 |
- KAPA, 0.02uM primer 1 tube of reaction: old F primer
Components | ==1 rxn== |
Circular DNA | 2.00 |
1uM AmpF6.4Sol | 1.00 |
1uM AmpR6.3Sol | 1.00 |
2x KAPA | 25.00 |
H2O | 21.00 |
Total volume (ul) | 50.00 |
Reaction codes
PCR conditions | Reaction code |
Phusion-NTC | NTC-P |
Phusion-old F primer-0.2uM primers | P-o |
Phusion-new F primer-0.2uM primer | P-n |
KAPA-NTC | NTC-K |
KAPA-old F primer-0.2uM primer | K-o |
KAPA-new F primer-0.2uM primer | K-n |
Phusion-old F primer-0.02uM | P-o1 |
KAPA-old F primer-0.02uM primer | K-o1 |
Program
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x25