You cloned an insert of approximately 3 kb into a single res

You cloned an insert of approximately 3 kb into a single restriction site within the multiple cloning site of a vector. The partial flanking sequences of the multiple cloning site (indicated in bold and underlined) and of the insert sequence in the desired orientation (in italics) are indicated below. You wish to use colony PCR on various clones to identify those which possess the insert in the correct orientation (as indicated below).

1. Which two primers could you use to identify clones which possess recombinant plasmids with the insert in the correct orientation?

Primers: (All sequences are written 5’ to 3’)

A. CGTTGCACAT B. AGCTCTGTGA C. AAATGGATTC

D. ATGTGCAACG G. GCAACGTGTA E. AGTGTCTCGA H. TCACAGAGCT F. TATGCCCATT

PLEASE ANSWER ALL

1500 3 000 3\'- CGTTGCACATTTAMAGGCCACGTGCE CTT TAC CTAAGTGCGAATGGGCATAACTGACCGAT AATGTTCCAT AGAGCTCTGTGA -5 Insert

Solution

In order to peform colony PCR and identify the insert, the primers should be corresponding to the flanking regions.

There the master mix should include reverse and forawrd primers M13F and M13R the forward and reverse primers, and do not add the Dna in this mix. Add enzymes at the end.

The amount of dna should be very less in order to get accurate results.

In colony PCR the Dna added are in colony form.

Add the small amount of colony at the PCR cold reaction.

AAATGGATTC primer should be used in order to the correct orientation.