Restriction Mapping Problem

[D.Smalley]

Hi guys, i was hoping someone might be able to point me in the right direction on a restriction mapping problem i have to do.

I have 2 plasmids named 18 and 18L. They are both clones from a pBluescript vector with a cDNA insert, inserted at the MCS. This region was restricted with Apa1 and EcoR1 and then put through an agrose gel separation. From this I have (i think) constructed a restriction map for both. Now, the EcoR1 site is supposed to be replicated at each end, but in the 18L plasmid one of them is missing and the MCS is also shorter: 18 is 5.8Kb and 18L is 5.0Kb with a 'missing' EcoR1 site.

I have to consider the relationship between these two plasmids. Being as they are the same vector and contain the same cDNA insert, my first thought is some sort of mutation? Or is it just that the cDNA has not been incorporated?

 

Any help is appreciated

[Greippi]

Often in the lab, you use restriction enzymes to cut your ligated plasmid to run on a gel, to make sure you have whatever it is you wanted to insert is there. If there's something like that going on, I'd say "aha! an incorrectly ligated vector!"

Do you know the size of the cDNA insert? If so, that will give you your biggest clue.

 

Mutation is extremely unlikely.

[D.Smalley]

Often in the lab, you use restriction enzymes to cut your ligated plasmid to run on a gel, to make sure you have whatever it is you wanted to insert is there. If there's something like that going on, I'd say "aha! an incorrectly ligated vector!"

Do you know the size of the cDNA insert? If so, that will give you your biggest clue.

 

Mutation is extremely unlikely.

 

Do you mean the size from the gel separation? Because by adding up the fragments I got 5.8 for one plasmid and 5.0 for the one missing the duplicated EcoR1 site. But i think you are correct, it must be wrongly inserted as the cDNA is inserted into the EcoR1 site, which means it should be duplicated, appearing at both ends.

[Greippi]

Yeah, I mean agarose gel DNA electrophoresis. Sometimes when you put an insert into a vector it doesn't work. So you gotta check all the fragments are of the right size on the gel.