cut-delete-insert once more - just cloning? - (Sep/28/2006 )

Hello!
And the problem is ... I have to cut my insert out of the vector (two restriction enzymes). Using next restriction enzyme I have to generate deletion in the fragment mentioned above. Having two bands I have to religate them and re-insert into vector. Sounds very nice, but ... it does not work in my hands.

Should I use dephosphorylation (SAP) at any step?
Should I dephosphorylate 'insert' before generating deletion?
After which steps should I purify DNA?
What controls would you prefer?

The more advices the better.

Thanks for your help .
Biolog2

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One question, how large are each of the fragment sizes, and how large is the deletion? Can you do a colony PCR to screen for the deletion?

I would dephosphorylaye the final vector which the inserts would go into.

Dephohorylation of the insert would not be a good idea. It would be counter productive as the final vector would then have to be phosphorylated to accept the religated insert. (The ends made by the 2 restriction enzyme digest would be dephosphorylated.) Of course you could PNK those ends, but the more steps involved the higher the danger of failure.

If the sizes of the sawn in half inserts are easy to discriminate from the original host vector and the discarded bit of DNA easily visualised, I do see any reason not to run all three digest together. (assuming all the enzymes can live together)

I would then gel purify this mix. Making sure the DNA is very well spread out, so as not to be too concentrated, which may pull down unwanted company.

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Hello!
Fragment sizes: ~1,5kb and 2,5kb (the rest of the plasmid which final ligated fragment is to be inserted to ~2,3 kb)
I cannot do colony PCR.
One of three enzymes cannot live together with rest of them.

What solution is the best now?
Thanks,
biolog2

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Date review
Original vector = ???bp
Half Insert 1 = 1.5kb
Half Insert 2 = 2.5kb
Bit left out = ???bp
Final vector = 2.3kb

Not every enzyme can live together... hmm... If the orignal vector is lets say 3kb (easily identifiable), it is still possible to do all the digest together. Do the lost salt digest first, once completed, proceed with the high salt digest by adding the high salt buffer to the digestion mix.

However if the digestion fragment can not be easily distinguished from each other (ie original vector produces fragments 2.4 or 2.3kb) then it would be simpler to cut out the insert, purify it, then cut it in half in a second digestion.

The final vector, onces it is opened, should also be gel purified to remove denatured plasmids. Denatured plasmids are plasmids in a configuration that is resistent to RE cutting but will transform rather well.


No colony PCR? That puts a dent in the bandwagon.

Does the Final vector have colour testing, someway to distinguish empty vectors from ones with inserts?

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