PCR Components For each component besides water briefly desc

PCR Components:

For each component besides water, briefly describe the component’s role in the PCR reaction.

PCR amplification recipe (stock concentrations):

14.4 L high quality nuclease-free water

6.0 L 5x Phusion HF buffer

3.0 L dNTPs (2.5 mM each of four dNTPs – dATP, dCTP, dGTP, dTTP)

0.3 L Phusion DNA polymerase

0.3 L pCD122 (a plasmid that serves as template DNA)

3.0 L sense primer CD474 (5 M)

3.0 L antisense primer CD475 (5 M)

30.0 L total reaction volume

Solution

1. 14.4 L high quality nuclease-free water : This is used to make up the final volume of the reaction mixture.Also, since it is deionised water free of DNase,RNase and nuclease,it prevents degradation of DNA.

2.6.0 L 5x Phusion HF buffer : This buffer contains MgCl2 that helps in annealing of the primer and denaturation of the template and also acts as a cofactor for the polymerase thus influencing the activity of the enzyme polymerase.It is used for high fidelity of the reaction.

3.3.0 L dNTPs (2.5 mM each of four dNTPs – dATP, dCTP, dGTP, dTTP : These act as building blocks that are used by the polymease to synthesize a new strand.

4.0.3 L Phusion DNA polymerase : It helps in in the synthesis of new complementary strand from the template strand by using the dNTPs.

5.0.3 L pCD122 (a plasmid that serves as template DNA) : This acts as the base strand whose complementary strand has to be made.

6.3.0 L sense primer CD474 (5 M) : This is the forward primer or the 5\' primer that binds to the first few nucleotides i.e 18-20 nucleotides of the sense DNA strand to initiate new DNA synthesis.

7.3.0 L antisense primer CD475 (5 M) :This is the reverse primer or the 3\' primer that binds to the last few nucleotides i.e 18-20 nucleotides of the anti sense DNA to limit the DNA synthesis.