Hi,
I'm extremely frustrated with this whole cloning business because my desired insert is not ligating into my vector backbone. I'm trying to clone a short microRNA (~150 bp) into my vector and it just refuses to go in. I've tried heating the plasmid vector for 5 minutes to get rid of any secondary structure before adding all my ligation reagents, and it still doesn't work. I've picked many clones and all of them return with the plasmid self-religating. I know for sure that the 2 restriction enzymes I'm using are working fine so I don't quite understand why it's self-religating. My next option is to treat the cut plasmid with CIP and see if that works out at all... if anyone has any ideas I'd be most grateful.
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#2
pcrman
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Posted 19 May 2011 - 07:26 PM
Quick fact check: Did you add extra bases outside the restriction site in your primers?
CIP your vector will help you get rid of most self-ligation. It will also help if you first do a TA cloning of your PCR product and then subclone the insert into an expression vector.
CIP your vector will help you get rid of most self-ligation. It will also help if you first do a TA cloning of your PCR product and then subclone the insert into an expression vector.
#3
moerae
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Posted 22 May 2011 - 03:38 PM
pcrman, on 19 May 2011 - 07:26 PM, said:
Quick fact check: Did you add extra bases outside the restriction site in your primers?
CIP your vector will help you get rid of most self-ligation. It will also help if you first do a TA cloning of your PCR product and then subclone the insert into an expression vector.
CIP your vector will help you get rid of most self-ligation. It will also help if you first do a TA cloning of your PCR product and then subclone the insert into an expression vector.
I've cut my miRNA from an existing vector.
