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Separating cut/non-cut vector - (Jan/13/2011 )

Hi all

I'm having a lot of trouble with this, hopefully there's an easy solution I'm not thinking of...

I am cutting open a vector to insert a gene of interest. Unfortunately, there are so few restriction sites I am able to use due to most of them being present in my GOI (using them would chop up my GOI too). So I have settled on two - MluI and NotI. I have PCR'd my GOI with these primers so it should be as easy as cutting opening up the vector and ligating them in. Or so I thought.

Unfortunately, these two restriction sites lie approx. 10bp's away from each other in the vector. Therefore, when I cut and run the resulting DNA on a gel, I cannot separate between cut and uncut vector. I was hoping that this wouldn't be a problem and that it would just all be cut. It wasn't. I attempted to ligate my gene into what I thought was cut vector. After doing 24 minipreps from the XL-1 blue that grew, not a single one of them holds the insert. In another case with another GOI using the exact same sites I got one sucessful prep out of 24.

I use CIP, but this obviously isn't going to work if the vector isn't cut open in the first place. Not surprisingly, on a CIP control plate (where no colonies should have grown) it was full of colonies.

I must be missing something here. It's obviously cutting a bit, as I got one successful prep. But god 1 in 24 is not a good statistic. I need help!

Thanks

-andyg2886-

NotI can be a tricky enzyme :)
First of all supercoiled plasmid is cut poorly ...always consider the things NEB is offering ...here is the link! ...see "Notes"!
Another problem with Not is that it needs 8nt upstream the restriction site to work ...i dont know your detailed sequence but consider this as well ...details see here.

I would try a sequential digest starting with NotI.

If you consider these points i think the cloning should be possible!

Good luck!
Best regards,
p

-pDNA-

I've heard many times it can be tricky!

Maybe it would be best to cut first with MluI as that seems to cut supercoiled ok (or NEB doesn't say otherwise). Then cut with NotI. But then of course the problem is they are only 10bp apart from each other so NotI might not then be able to work.

So maybe start with NotI but use 5x fold? I've never done anything like this before. Maybe you could help me. My normal reaction is this:

1 ug DNA (5ul)
10x NEbuffer (3 ul)
NotI (1 ul)
Water (21 ul)

Total 30 ul reaction

Incubate for 2hrs at 37 degrees. If I use 5x fold enzyme, what amount of everything else should I use?

Cheers

-andyg2886-

so 1 L enzyme contains 10 U ...normally 1 L is sufficient to cleave 1 g.
For 1 g i would use at least a 50 l reaction volume!!!

In the case of NotI and supercoiled DNA you can use e.g. 2.5 l enzyme ...i would not use 5 l in 50 L that is too much for that volume!
Incubate for ~8h.

Regards,
p


andyg2886 on Thu Jan 13 21:21:21 2011 said:


I've heard many times it can be tricky!

Maybe it would be best to cut first with MluI as that seems to cut supercoiled ok (or NEB doesn't say otherwise). Then cut with NotI. But then of course the problem is they are only 10bp apart from each other so NotI might not then be able to work.

So maybe start with NotI but use 5x fold? I've never done anything like this before. Maybe you could help me. My normal reaction is this:

1 ug DNA (5ul)
10x NEbuffer (3 ul)
NotI (1 ul)
Water (21 ul)

Total 30 ul reaction

Incubate for 2hrs at 37 degrees. If I use 5x fold enzyme, what amount of everything else should I use?

Cheers

-pDNA-

Ok I have cut with Not I as described above and extracted the bands.

What conditions should I now use to cut with MluI?

Thanks.

-andyg2886-

You're going to have trouble cutting your PCR product with NotI as well -- it needs a lot of bases 5' to its recognition site to cut efficiently. When we have to add this site to the 5' end of a primer, we TA clone the PCR product first, then recover the band from the double-digested TA vector to insure efficient cutting.

-HomeBrew-

the ones suggested by the manufacturer of the enzyme ...to my knowledge MluI should not be that tricky as NotI is (but i have to confess that i never used MluI)! also consider the comment of Homebrew ...thats really a good advice!

And next time ...if possible avoid NotI :) ...its a pain in the neck!

Regards,
p

andyg2886 on Fri Jan 14 14:03:10 2011 said:


Ok I have cut with Not I as described above and extracted the bands.

What conditions should I now use to cut with MluI?

Thanks.

-pDNA-