Noi/NOTES/2011-9-1: Difference between revisions

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* Received 4 new primers for this experiment
* Received 4 new primers for this experiment
** AmpF7NU.Sol
** [[Media:AmpF7AUSol_Seq.pdf| AmpF7AUSol]]
** AmpF7AU.Sol
** [[Media:AmpF7NUSol_Seq.pdf| AmpF7NUSol]]
** Syb_FP5A
** [[Media:Syb_FP5A_Seq.pdf| Syb_FP5A]]
** Syb_RP7
** [[Media:Syb_RP7_Seq.pdf| Syb_RP7]]
'''Tests:'''
'''Tests:'''
* Does KAPA work?
* Does KAPA work?
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[[File:2011_09_01_test-tagging-primer-qPCR-2.png| 600px]]<br>
[[File:2011_09_01_test-tagging-primer-qPCR-2.png| 600px]]<br>
* 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.  
* 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 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. In addition, the old F primer showed higher efficiancy than the new one with uracil residue.
* 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 (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.
* 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: [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2011-9-2#PCR_set_up]]

Latest revision as of 00:16, 1 June 2012

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]]