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Release of an insert from the cloning vector using restriction enzymes - (Jun/24/2008 )

Hi guys,

I have fluorescent PCR products that I use in TRFLP.
This is what I want to do with them :
-reamplify these amplicons with the same NON-fluorescent primers
-clone the non-fluo amplicons (pGEM T easy vector probably)
-release the insert (1.5bp) from the vector with restriction enzymes
-reamplify the insert with the fluorescent versions of the primers and do TRFLP on each of them
-put the restriction digests on an automated sequencer to screen the clones (I am looking for those clones that give a particular restriction fragment in TRFLP)
-finally, sequence the clones that actually contain the sequences giving the restriction fragment I am looking for

First of all, does that look duable ?
Second, I am concerned about the use of RE in step #3 (release) because I do not know the sequence of the inserts (they are environmental sequences). I have no idea if these sequences have or not the restriction sites cut by the enzymes I will use for the release.
So my second question is : how could I release the insert without using RE ? Could I PCR amplify it and cut off the corresponding bands from the gel ?

thanks !

-valou-

QUOTE (valou @ Jun 24 2008, 11:00 AM)
Hi guys,

I have fluorescent PCR products that I use in TRFLP.
This is what I want to do with them :
-reamplify these amplicons with the same NON-fluorescent primers
-clone the non-fluo amplicons (pGEM T easy vector probably)
-release the insert (1.5bp) from the vector with restriction enzymes
-reamplify the insert with the fluorescent versions of the primers and do TRFLP on each of them
-put the restriction digests on an automated sequencer to screen the clones (I am looking for those clones that give a particular restriction fragment in TRFLP)
-finally, sequence the clones that actually contain the sequences giving the restriction fragment I am looking for

First of all, does that look duable ?
Second, I am concerned about the use of RE in step #3 (release) because I do not know the sequence of the inserts (they are environmental sequences). I have no idea if these sequences have or not the restriction sites cut by the enzymes I will use for the release.
So my second question is : how could I release the insert without using RE ? Could I PCR amplify it and cut off the corresponding bands from the gel ?

thanks !



please, I need your advices :-))))))

-valou-