1 Describe what result you will expect to get from a PCR whe

1. Describe what result you will expect to get from a PCR when the template is:1) a recombinant pET41/EGFP; 2) a non-recombinant pET41 plasmid? 2. indicate what the negative and positive control reactions for your PCRs will be, and the reason for inculuding this reaction.

Solution

For any PCR reaction to occur, it is essential to have one pair of primers ( forward primer and reverse primer) along with other components for the PCR reaction.

pET41 is a plasmid of 5933 bp whereas the size of EGFP is 720bp. If we design a primer that gives the amplified PCR product for the recombinant of pET41/EGFP the size will be 5933+720=6653bp

where as non recombinant pET41 will have PCR product of size 5933bp.

For the above reactions the controls that can be taken are:

Negative control for the reaction will have all the PCR comonents except the DNA template( in this case the plasmids), this will show that the primers used for amplifying the Recombinant and non-recombinant are specific in nature. We can also use any other known plasmis (say puc 18) as negative control to show that the primers for pET41 are specific. the purpose of negative control is to make sure that the primers are specific and their is no contamination present in PCR components.

Positive controls are always the samples whose results has been established. We can also use any other known plasmids (say puc 18) as positive control , the purpose of the positive control is to make sure that the PCR reaction is correct and is taking place and other component of PCR master mix like DNA polymerase, PCR buffer, dNTPS are functioning properly during the reaction.