Consider the following information to answer questions 1014

Consider the following information to answer questions 10-14: 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.

1. Which restriction site would you add to your forward primer?

2. Which restriction site would you add to your reverse primer?

3. 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 SalI. What fragment sizes would you expect in each of the two orientations?

a. Correct orientation:

b. Incorrect orientation:

HELP WITH ALL PLEASE

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

Solution

Answer -Restriction site I add in forward primer would be E(EcoRI)

Restriction site I add in reverse primer would be B (BamHI)

3) 2 fragmens size 0.5kb and 5.1 kb