Question

3) How can you use PCR to clone a 2000bp gene into a plasmid vector?

Answer 1

PCR based cloning is different from the traditional method in which gene of interest and vector both are amplified using polymerase chain reaction (PCR). Both the gen of interest and vector are ligated without the use of restriction enzymes. This process is advantageous than traditional method being rapid and can clone gene fragments that are not available in large amounts.

The following steps are involved in the PCR based cloning method:

1. Designing of primers for PCR based cloning

Primer design for pCR based cloning should consists of; Leader sequence, Restriction site and Hybridization sequence.

Leader sequences are 3-6 base pairs on the 5’ end of the primer

Restriction site (6-8 bp) depends on the individual doing an experiment. This should be determined precausiously as it should not cut within the gene of interest and must be present at desired location of the vector (multiple cloning site).

Hybridization region (18-21 bp) is the sequence of primer that binds to the gene sequence to be amplified.

2. PCR reaction is carried out to amplify the gene of interest using high fidelity taq polymerase for reducing mutation

3. Isolation of PCR product from the PCR reaction

4. Preparation of DNA digest and vector plasmid using restriction enzymes

5. Isolation of gene and plasmid vector by gel purification technique so as to have a desired gene size

6. Ligation of gene of interest into the vector

Typical DNA ligation is required to insert the gen of interest into the plasmid vector

7. Transformation of ligated plasmids into competent cells like DH5α. The number of colonies obtained is the indication of good transformation

8. Isolation of plasmids with gene of interest using overnight grown cultures. Bacterial colonies picked from the transformed plates will be grown and DNA purification is carried out on DNA using restriction enzymes that were used earlier. The product is run on agarose gel and provide you two bands, vector and insert.