You want to clone geneX. To do this, you want to ligate the ORF of geneX into pl

Question

You want to clone geneX. To do this, you want to ligate the ORF of geneX into plasmid Y. Plasmid Y contains the sequence (5'-AAGCTT-3') which is the recognition site for the restriction endonuclease HindIII. Since geneX sequences don't contain the HindIII recognition sequence, you first amplify the ORF of geneX with primers that contain the restriction endonuclease site for HindIII with PCR. You then digest your PCR products, purify the PCR product by gel electrophoresis and EtOH precipitation and then ligate the insert and the plasmid. But you find out that your experiment did not work (the ligation did not work). You realize that you forgot to purify your PCR product before you added HindIII. Why should the lack of purification of your PCR product at that step interfere with the later steps? Please explain briefly.

Explanation / Answer

If you forget to purify the PCR product, the Taq polymerase can’t be removed. The enzyme is heat stable and can’t be heat deactivated also. This enzyme has the property to fill the 5’ or 3’ overhangs in a double stranded DNA. So, ultimately the double stranded DNA will not be sticky but blunt ended. In our case the sticky ends generated by Hind III will create 5’ overhangs which will be refilled by the presence of DNA polymerase in unpurified PCR product and hence no ligation can be performed with vector cut with same restriction enzyme.

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