Noi/NOTES/2011-9-1

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Randomly tagging primer test[edit]

Idea
Athurva's idea:...........will be added later

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]]