Hello all, I have absolutely no clue how to start this problem and I would like to have some cues or just something to chew on...thank you!!

I know what a plasmid is , I know what sticky ends (cohesive extremities) are but I dont know how the multiple cloning sites in this plasmid are located so im a bit lost!

 

Question: U must clone a DNA fragment having Sau3A sticky ends.

The plasmid to be used for the cloning experiment has the following MCS

EcoR1, BamH1, Xba1, HindIII

 

*Describe how you would do the cloning experiment.

 

Restriction enzyme recognition sites

EcoR1 = GAATTC (the enzyme cuts after the G)

BamH1 = GGATTC (the enzyme cust after the first G)

Xba1 = TCTAGA (the enzyme cuts after the first T)

HindIII = AAGCTT (the enzyme cuts after the first A)

Sau3A = GATC (the enzyme cuts before the G)

The location or even the precise sequence of the MCR is quite irrelevant, they are constructed that each of the enzymes will cut only once. The more important question is how the overhang looks like for Sau3a looks like and search for compatible ends.

The location or even the precise sequence of the MCR is quite irrelevant, they are constructed that each of the enzymes will cut only once. The more important question is how the overhang looks like for Sau3a looks like and search for compatible ends.

 

Thanks for your help CharonY. So since sau3a cuts beofre g, i believe my overhangs would be

5- (space) GATC-3

 

3-CTAG (space) -5

 

But i dont understand how this helps me to solve the problem...

Edited October 3, 201213 yr by boa