Question

M 1 2 Figure 3. Agarose gel electrophoresis (1%) of restriction enzyme digested plasmid and PCR product to confirm presence of the B-glucuronidase gene in PET28a plasmid

Two experiments were performed in order to confirm the Beta-glucuronidase gene from E. coli was present in the pET28a plasmid. (Lane1) PCR was performed to amplify the Beta-glucuronidase gene and if the gene was present then a single band would appear at ~7,300 bp. However, the band appeared to drag down the gel but stopped ~7,300 bp. What would cause the PCR product to smear down the gel and does this mean that the amplification of the PCR product was successful or unsuccessful? (Lane 2) The pET28a plasmid was digested using the restriction enzyme AluI. If the digest was successful, two bands would appear at ~85 bp and ~6500 bp. However, it looks like there are 4 bands which appeared. Why would a single enzyme digest cause 4 bands to appear on the gel?

Answer 1

A) gene specific pcr showed smear in gel which could be cause of DnaseI or some salt contamination which is degrading vector backbone and hence it's showing smaer of DNA.kindly use fresh reagent for pcr and run positive and negative control.

B) digestion with Alu I restriction enzyme is not giving expected result which means that Alu site is present in back at multiple point or there is some contamination in vector ligation.

I think you used different restriction enzyme combination to check the releases or to confirm cloning of right gene in right direction. Or final solution is that you can send the clone vector of sequencing to check cloning of gene.

Hope this will help you..all the very best !!!