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

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= Randomly tagging primer test =
= Randomly tagging primer test =
* Received 4 new primers for the project
'''Idea'''<br>
** AmpF7NU.Sol
Athurva's idea:...........will be added later<br>
** AmpF7AU.Sol
 
** Syb_FP5A
* Received 4 new primers for this experiment
** Syb_RP7
** [[Media:AmpF7AUSol_Seq.pdf| AmpF7AUSol]]
** [[Media:AmpF7NUSol_Seq.pdf| AmpF7NUSol]]
** [[Media:Syb_FP5A_Seq.pdf| Syb_FP5A]]
** [[Media:Syb_RP7_Seq.pdf| Syb_RP7]]
'''Tests:'''
'''Tests:'''
* Does KAPA work?
* Does KAPA work?
* Does new F primer work for both Phusion and KAPA?
* Does new F primer (contains uracil) work with both Phusion and KAPA?
* Can we reduce primer conc. From 0.2uM to 0.02uM?
* Can we reduce primer conc. From 0.2uM to 0.02uM?
== PCR set up ==
== 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'''
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''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<br><br>
* '''Phusion, 0.2uM primer, 3 tubes of reactions : 1. old F primer (AmpF6.4.Sol), 2. new F primer (AmpF7AU.Sol), 3. NTC'''<br>
* Note: NTC used old F primer (AmpF6.4.Sol)
{| {{table}} border = 1
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Components'''
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|}
|}
* aliquot 47ul, add 1ul of 10 uM F primer and 2ul of circular DNA<br><br>
* aliquot 47ul, add 1ul of 10 uM F primer and 2ul of circular DNA<br><br>
* '''KAPA, 0.2uM primer, 3 tubes of reactions: 1. old F primer (AmpF6.4.Sol), 2. new F primer (AmpF7AU.Sol), 3. NTC'''
* '''KAPA, 0.2uM primer, 3 tubes of reactions: 1. old F primer (AmpF6.4.Sol), 2. new F primer (AmpF7AU.Sol), 3. NTC'''<br>
* Note: NTC used old F primer (AmpF6.4.Sol)
{| {{table}} border = 1
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Components'''
Line 85: Line 110:
|-
|-
| Total volume (ul)||50.00
| Total volume (ul)||50.00
|}<br>
'''Reaction codes'''
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''PCR conditions'''
| align="center" style="background:#f0f0f0;"|'''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
|}<br>
|}<br>
'''Program'''<br>
'''Program'''<br>
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x25<br>
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x25<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.
* 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: [[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]]