restriction digestion problem... - (Oct/17/2005 )

Hi all....
I double digested my vector (4.7kb) with EcoR I/NheI...
it supposed to yield 750bp and 3950bp band.....

But, after I run it on 0.8% gel I saw 2 band ....1 is 750 bp as expected but the other band is ~4-5kb and this band is a very bright band....
Is anyone know why it happened??

thx in advanced.....

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When you say you're digesting your vector, do you mean you're digesting your clone (i.e. are you popping out a 750 bp insert)?

If that is the case, what does vector alone, cut the same, way run like in comparison to your larger band?

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I run the digested vector with the undigested vector (I forgot to digest it only with 1 of R.enzyme in order to linearized it).....
The undigested vector have 3 band appear on the gel.....

I still wondering why if the GFP already popped out (there is 750bp band ) why I got ~4-5kb instead of ~3.9 kb ?.......For info I digested the vector for 1 hr.....

thx...

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hi
as you mentionned, do a single digestion to check the efficiency and the appearant lenghth of your single digested plasmid. That may answer your problem. Nhe I is a lazy enzyme. So 2h would be greater maybe?
Neb website tells that for an Nhe1 BamH& digestion, sequential events are required (table here)...
i'm currently using buffer 2 of NEB + BSA as they tell 100% cleavage efficiency in their table. I would recommend you not use the bamH1 buffer for the double digest.
fred

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That speaks for an undigested plasmid and what you see is the linear vector. That's why this band is fat cause only a small portion of your construct is cut. How intense is the insert band ? If it's adiscrete band it will be right with this hypothesis.

Anyway as Fred recommend do a single digest with each enzyme and incubate a bit longer 2h is not so luxous for a plasmid digestion !

pesji

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The ~4kb band should be much brighter than the 750bp band and depending of how much DNA you have in your inicial prep the larger band may be so intense that expands from 4-5Kb (very close in aparent size to your expected 3950bp).
I am not sure what do you need that DNA for but if you need the 750bp insert for cloning, just Gel purify it and forget about the rest of the vector.
I hope it helps
Clarice

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Hii alll thx for the input...=)
The insert band is very bright .....
my R.enzyme is NheI/EcoRI not BamHI and I have done double digest with NheI/EcoRI several times and it work fine....

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Hi clarice...thx for the reply...
In this case I need the vector not the insert ...

However, I already gel purify the vector (~4-5kb) .....

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if you see a bright strong band about 4-5kb, I bet it's a mixture of single-and double-cut

some is vector + insert (single cut)
some is vector alone (double cut)

band would be bright anyway, but if it were a tight doublet (and there isn't much difference between 3.9 and 4.6) it might appear as a whopper a little over 4?

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Hi aimkins...thx for the reply....
I think you're right maybe it because some of the vector is double digeted and some were not....
I already gel extract this band ....I will use this digested vector for ligation with my gene of ineterest...do you think it still can work for ligation eventhough the band is the mixture of the digested vector and the undigested one?

thx a lot

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