Question

You have embark on a genomic library construction project. What is the strategy that you will be taking to ensure successful cloning of genomics inserts into the pGLO vector - you are well acquainted with this plasmid molecule? You have been provided with a map of the pGLO plasmid.

a) Where in the plasmid vector molecule would you insert the genomics fragments that you isolated and what restriction enzymes would you choose?

b) how would you determine that indeed you have recombinants – that is, genomics inserts ligated successfully into the pGLO cloning vector?

Answer 1

Q1 Answers

Plasmid map of pGLO has shown below. Gene of interest should be inserted in the multiple cloning sites. This site contain restriction sites for many restriction enzymes. Depending upon the availability, any pair of enzymes can be used. For e.g. here i am mentioning to use of SphI and EcoR1 for the Insertion of DNA into vector.

Q2 Answers

There are two methods to confirm this.

1. Plasmid sequencing: In this method primers for the gene of interest will be used to amplify the construct and nucleic acid sequence of the vector containing gene of interest will be determined. Then, you can match sequence of the gene of interest with the result of plasmid sequencing to confirm gene of interest has inserted in the vector.

2. Digest the vector having gene of interest with the same restriction enzymes used for the cloning. Vector without any insert DNA will be also digested with the same restriction enzymes. Run both the samples along with DNA ladder on agarose gel electrophoresis. compare the bands in both the samples and also check the presence of any band corresponds to the molecular weight of gene of Interest to confirm vector contains gene of interest.