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Quy trình chạy PCR

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1 Quy trình chạy PCR on Tue Oct 13, 2015 9:15 am

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[*]Prepare the following 50 µl reaction in a 0.5 ml PCR tube on ice: 

* Due to the difficulties in pipetting small volumes of enzyme, Taq DNA Polymerase can be diluted in 1X reaction buffer. For example, 1 µl of Taq DNA Polymerase is mixed with 4 µl of 1X reaction buffer and 1 µl of that mixture is added to the reaction. Enzyme diluted 1X reaction buffer should not be stored for future use.








Component25 μl reaction50 μl reactionFinal Concentration
10X ThermoPol or Standard Taq Reaction Buffer2.5 µl5 μl1X
10 mM dNTPs0.5 µl1 μl200 µM
10 µM Forward Primer0.5 µl1 μl0.2 µM (0.05–1 µM)
10 µM Reverse Primer0.5 µl1 μl0.2 µM (0.05–1 µM)
Template DNAvariablevariable<1,000 ng
Taq DNA Polymerase*0.125 µl0.25 µl1.25 units/50 µl PCR
Nuclease-free waterto 25 µlto 50 µl 

[*]Gently mix the reaction and spin down in microcentrifuge. 
If the thermocycler does not have a heated cover, add one drop of mineral oil to the reaction tube to prevent evaporation.


[*]Cycling Conditions for a Routine PCR:





CYCLE STEP 
TEMP 
TIME 
CYCLES
Initial Denaturation 95°C 30 seconds 
Denaturation
Annealing
Extension 
95°C
45-68°C
68°C 
15-30 seconds
15-60 seconds
1 minute per kb 
30
Final Extension72°C 5 minutes 1
Hold4°C  
Twisted Evil
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