Question

8. Draw a pipetting scheme for your PCR reactions. Indicate how much of what will go into each tube. Make sure to include the correct number of PCR reactions. (4 points)

Answer 1

PCR also called as Polymerase Chain Reaction is a technique that has been developed to amplify the amount of DNA (gene of our interest). Here, the components required include initially a DNA template (our sample), Taq polymerase that can perform polymerization, primers to initiate strand synthesis ( both forward and reverse primer), the four nucleotides (dATP,dCTP,dGTP,dTTP) and nuclease free water.

The concentration of different components of a PCR reaction mixture is as following:

COMPONENT	25 $\mu$ l reaction
10X Standard reaction buffer	2.5 $\mu$ l
Template DNA	Varies
Taq DNA Polymerase enzyme	0.125 $\mu$ l
10 $\mu$ M Forward primer	0.5 $\mu$ l
10 $\mu$ M Reverse primer	0.5 $\mu$ l
10 mM dNTP's	0.5 $\mu$ l
Nuclease free water	to 25 $\mu$ l

Since it is is difficult to pipette small volumes of PCR reaction mix, it is good to prepare reaction mix for the total number of samples and then divide them equally to each of the sample.

The different steps in a PCR reaction includes, Denaturation, Annealing and Extension. The time required and the number of cycles for each step is as follows :

STEPS	TEMPERATURE	TIME	NO. OF CYCLES
Initial Denaturation	950c	30 seconds	1
Denaturation Annealing Extension	950c 45-680c 680 c	15-30 seconds 15-60 seconds 1minute/ Kb	25- 30 cycles
Final Extension	72 0c	5 minutes	1
Hold	40c