Noi/NOTES/2011-9-1

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Revision as of 15:42, 2 September 2011 by >Noi (→‎PCR set up)
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Randomly tagging primer test

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

  • Received 4 new primers for this experiment
    • AmpF7NU.Sol
    • AmpF7AU.Sol
    • Syb_FP5A
    • Syb_RP7

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

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, but the efficiency was lower than Phusion. I was think that if we use two-step PCR at 60C or extension at 60C, would we see higher amplification?
  • For the reaction with lower primer concentration (0.2uM), the curve slightly went up after 20 cycles, but the was no NTC, so it's hard to say if it's the real amplification.