Noi/NOTES/2011-9-1
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Randomly tagging primer test[edit]
Idea
Athurva's idea:...........will be added later
- Received 4 new primers for this experiment
Tests:
- Does KAPA work?
- Does new F primer (contains uracil) work with both Phusion and KAPA?
- Can we reduce primer conc. From 0.2uM to 0.02uM?
PCR set up[edit]
PCR conditions |
1. Phusion-NTC-old F primer-0.2uM primers |
2. Phusion-old F primer-0.2uM primers |
3. Phusion-new F primer-0.2uM primers |
4. KAPA-NTC-old F primer-0.2uM primers |
5. KAPA-old F primer-0.2uM primers |
6. KAPA-new F primer-0.2uM primers |
7. Phusion-old F primer-0.02uM primers |
8. KAPA-old F primer-0.02uM primers |
- Circular DNA: pooled BSPP captured DNA of blood samples from UCLA
- 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 |
Program
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x25
File:2011 09 01 test-tagging-primer-qPCR-2.png
- From the realtime-PCR curve, Phusion with old F primer at the concentration 0.2uM showed highest amplification efficiency, but can not amplify the new F primer containing uracil.
- KAPA works with both old F and new F primers at the concentration 0.2uM, but the efficiency was much lower than Phusion. I think that if we adjust the condition to be optimal for KAPA, we might be able to increase the amplification efficiency for the future experiment. In addition, the old F primer showed higher efficiancy than the new one with uracil residue in KAPA.
- For the reaction with lower primer concentration (old F primer, 0.02uM F/R primers), the curve slightly went up after 20 cycles in both Phusion and KAPA, but there was no NTC, so it's hard to say if it's the real amplification.
Comments
- Dr. Zhang suggested that I cannot compare the absolute intensity between the two different conditions since the amount of SYBR Green I in different kits are different. The Ct values should be used to account for the efficiency of the enzymes from different companies.
- By looking at the Ct values,
- The Ct value of reaction #2 ( Phusion-old F primer-0.2uM primers) and #5 (KAPA-old F primer-0.2uM primers) were pretty much the same ~ 18.2. Whereas the Ct value of reaction #6 (KAPA-new F primer-0.2uM primers) was 19. This imply that with the same old F primer, KAPA has the same efficiency as Phusion .
- Continued on 2011_09_02: [[1]]