T A cloning problems (View forum version)



rob180

Posted 24 August 2009 - 03:26 AM

Hi,
I am new to cloning and have just started cloning sequences using the AccepTor vector TA cloning kit. I am attempting to perform a reaction using ~200b.p. fragments that have been generated using four different primer pairs. The cloning appears to be working successfully and have been getting numerous transformed colonies. However, I have been performing PCR amplification on the colonies afterwards using the different primer pairs to determine which fragment has been taken up by which colony and it appears that some colonies contain more than one fragment. I have been told that this may have occured due to different fragments ligating together during the cloning reaction through the action of the ligase and that dephosphorylation of the fragments prior to the reaction may prevent this from happening.


I would be grateful for any opinions on this.

HomeBrew

Posted 24 August 2009 - 05:05 AM

It would be easier to clone each PCR fragment in a separate ligation reaction. Dephosphorylation of the PCR products would presumably work as well, but this frequently introduces its own set of problems (e.g. damaged ends).

rob180

Posted 24 August 2009 - 08:15 AM

It would be easier to clone each PCR fragment in a separate ligation reaction. Dephosphorylation of the PCR products would presumably work as well, but this frequently introduces its own set of problems (e.g. damaged ends).

Hi,
I would clone each loci seperately but the kit that I'm using doesn't have reagents for that many reactions and I am unable to afford another one.

Warren

Posted 03 September 2009 - 11:20 PM

It would be easier to clone each PCR fragment in a separate ligation reaction. Dephosphorylation of the PCR products would presumably work as well, but this frequently introduces its own set of problems (e.g. damaged ends).

Hi,
I would clone each loci seperately but the kit that I'm using doesn't have reagents for that many reactions and I am unable to afford another one.

there is no need to dephosphorylate PCR products unless you specifically phosphorylated the primers prior to PCR (which would make no sense here), primers generally do not have a 5'-phosphate. And if your doing TA cloning, the A overhangs are not compatible anyway. Regardless you should be able to check the size of your insert by restriction digestion, independent of the PCR. There is something else to this story.....of the top of my head perhaps one of your inserts is able to be amplified by more than one of your primer sets? Its hard to say without more details....