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Inverse PCR ligation - (Nov/20/2008 )

Hi

I have just carried out an inverse PCR on a plasmid containing and insert (total size of plasmid + insert = 7kb). When i run the pcr prodcut on a gel i get a band at about 6kb which corresponds to my product and is about right. I cut out this band, purified it and done a blunt end ligation to re-circularize the plasmid. I set up 3 reactions for the ligation:
1. 100ng of pcr product + ligase and ligase buffer
2. 25ng of pcr product + ligase buffer
3. 25ng of pcr product with ligase buffer but no ligase.

I carried out these ligation reactions at 4C over night. The following day i carried out a transformation and got about 4 colonies from reaction 1, 7 colonies from reaction 2 and 10 colonies from reaction 3 (which had no ligase). Surely i should have got the most number of colonies for reacttion 1 and no colonies for reaction 3. Could someone please try to tell me what might have happened? Is it worth picking the colonies and getting the plasmids seqeunced or should i just start again from scratch?

Thanks

-draj2k-

not sure what happened,maybe pure bad luck that this specific transformation was more succesful, the difference between 7 and 10 isn't that big.

Were your primers phosphorylated? (needed to enable ligation).
Apart from this: you could DpnI digest your PCR reaction, kills the parental plasmid as for site directed mutagenesis (dna needs to be methylated for digestions, PCR product and plasmid is when using most e. coli strains), then go on with gel purification, should decrease background massively.

Pick some colonies and do restriction digest to check the length, then consider sequencing.

QUOTE (draj2k @ Nov 20 2008, 05:56 AM)
Hi

I have just carried out an inverse PCR on a plasmid containing and insert (total size of plasmid + insert = 7kb). When i run the pcr prodcut on a gel i get a band at about 6kb which corresponds to my product and is about right. I cut out this band, purified it and done a blunt end ligation to re-circularize the plasmid. I set up 3 reactions for the ligation:
1. 100ng of pcr product + ligase and ligase buffer
2. 25ng of pcr product + ligase buffer
3. 25ng of pcr product with ligase buffer but no ligase.

I carried out these ligation reactions at 4C over night. The following day i carried out a transformation and got about 4 colonies from reaction 1, 7 colonies from reaction 2 and 10 colonies from reaction 3 (which had no ligase). Surely i should have got the most number of colonies for reacttion 1 and no colonies for reaction 3. Could someone please try to tell me what might have happened? Is it worth picking the colonies and getting the plasmids seqeunced or should i just start again from scratch?

Thanks

-vairus-