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vector auto ligation problem - (Mar/23/2005 )

Hi guys, my subclone insert into pGEX4T2 vectors. I picked up some colonies and did mini pre of plasmids, the products gave me positive PCR test result, so I did not do double digestion. But expression result gave me only GST protein. Any advice? I wonder why the PCR test is not dependable.


how do you know that it is only the GST being expressed?

is your DNA fragment in frame with the GST? is there a premature stop codon?

I'd still the do the double digest on your minipreps just to see if it is consistent with your PCR result.