- Your friend Silvester wishes to subclone a 1.7 kb DNA fragment into the pCR 2.1 TOPO plasmid vector. His goal is to amplify the desired fragment by...

6.- Your friend Silvester wishes to subclone a 1.7 kb DNA fragment into the pCR 2.1 TOPO plasmid vector. His goal is to amplify the desired fragment by PCR. At the end of the PCR cycling reaction, he immediately adds 4 μL of PCR reaction, (the maximum of DNA allowable) to the ligation mix. He did the transformation and plated it onto solid media containing the right amount of X-gal and ampicillin.

He picked a single white colony, inoculated it into liquid LB media with ampicillin and grew it up overnight. The next day he purified the plasmid DNA from the colony and sent it for sequencing. He prepared two tubes of DNA, one sample of plasmid will be sequenced using the M13 -20 primer to sequence the bottom strand. The second DNA sample will be sequenced with the M13 Reverse primer to sequence the upper strand. Silvester got the sequence back and the chromatograms were very good. When he BLAST'ed the sequences using NCBI he realized that he had cloned a PCR product that was only 800 bp. He was upset because he felt that since he'd used blue-white selection and chose a white colony that he should have isolated the fragment he was interested in. He is confused and does not understand his results.

(i) Why was Silvester able to conclude with confidence that the PCR product he submitted for sequencing was only 800 bp? (10 pts).

 (ii) Explain why a 800 bp sequence was ligated into the pCR 2.1 TOPO vector although his aim was to amplify a larger PCR product (5 pts).

    (iii) Give him some recommendations that would help to him to select the right clones for sequencing in the future (15 pts).