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= Randomly tagging primer test = '''Idea'''<br> Athurva's idea:...........will be added later<br> * Received 4 new primers for this experiment ** [[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:''' * 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 == {| {{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 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1 rxn''' | align="center" style="background:#f0f0f0;"|'''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<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'''<br> * Note: NTC used old F primer (AmpF6.4.Sol) {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1 rxn''' | align="center" style="background:#f0f0f0;"|'''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<br><br> * '''Phusion, 0.02uM primer 1 tube of reaction: old F primer''' {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''==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 |}<br> * '''KAPA, 0.02uM primer 1 tube of reaction: old F primer''' {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''==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 |}<br> '''Program'''<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]]
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