You are interested in the directional cloning of the maize h

You are interested in the directional cloning of the maize heat shock protein gene (2 kb) into the vector pGAL. You need to choose the restriction sites that you will include in your PCR primers and that you will use to cut the vector pGAL (5 kb). The following are maps of the multiple cloning site of pGAL and the DNA sequence that encodes the maize heat shock protein.

10. Which restriction site would you add to your forward primer?
11. Which restriction site would you add to your reverse primer?
12. You isolate plasmid DNA from a colony which you think has the insert of interest. To verify
your assumption, you digest the recombinant plasmid with XbaI. What fragment size (s) is
(are) expected?

To verify the orientation of the insert within the pGAL vector, you perform a digest on the
positive recombinant with SspI. What fragment sizes would you expect in each of the two
orientations?

13. Correct orientation:
14. Incorrect orientation:

PLEASE ANSWER ALL AND EXPLAIN THOUGHT PROCESS

GAL promoter E EcoRI S SspI E S Nc Nd- Ndel 3\' Nic- co X- Xbal pGAL multiple cloning site BJ BamHI 0.25 0.25 B 0.5 kb TAA 3\' 0.1 0.2 0.7 5\' ATG restriction map of the open reading frame encoding the maize (corn) heat shock protein

Solution

Directional cloning is a process in which the fragment of interest is inserted into the cloning vector in a particular direction. In it always two restriction enzymes are used so that the directionality can be maintained.

Again, the restriction sites which will be included in the forward and reverse primers would be basically those which are not present in the fragment of interest so that it doesnt gets cleaved, here the maize heat shock protein. The given restriction map of the open reading frame of maize heat shock protein doesnt contain the restriction sites for Nde1 and Nco1.

Hence for the forward primer restriction site would be for restriction enzyme Nde1 site will be 5\'CATATG3\' and 3\'GTATAC5\'. and for Reverse primer it would be for Nco1 which will be 5\' CCATGG3\' and 3\' GGTACC5\'.

On digesting with Xba1 only one fragment will appear beacause there is only one restriction site for it. Fragment will be of 2kb( fragment) + 5kb (vector).

Digestion will only take place if the insert is in correct orientation that will be two fragments one of the insert and other for the vector. The fragment will not ligate in the wrong direction since there\'s no ligation sites . thats the essence of directional cloning.

correct orientation will be in direction 5\' to 3\'.