6. You want to clone the Xl ORF into the pQE3O plasmid. To do this, you find the

Question

6. You want to clone the Xl ORF into the pQE3O plasmid. To do this, you find the X1 MRNA (eDNA) sequence from NCBI which is shown below (only the beginning and the end of the ORF is shown). and you design the PUR primers, which are also shown below The forward primer contains a restriction endonuclease site for BamHI (GGATCC), and the reverse contains the site for Hind III(AAGCTT); Using these PCR primers, you perform RT-PCR. Then you purify your PCR product, digest it with BamHl and Hind II. You also will digest the pQE3O plasmid with the same enzymes and clone the PUR product into the plasmid. However, you realize that there are 3 pQE3O plasmids (pQE3O, -31, Please answer the following questions based on the infuriation given above. a. Your forward primer does not contain the start codon. Why? Please explain briefly. b. Which of the three different pQE vectors is suitable for cloning the X1 gene amplified by your primers? Please explain briefly.

Explanation / Answer

a. Here we are cloning an ORF, so, cloning to be done from start codon ATG to stop codon TAA. Therefore we have to add GGATTC (BamH1) to forward primer and reverse primer contains the site for AAGCTT (HindIII).

b. PQE -31 is suitable vector for cloning the X1 gene amplified by the primer.

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