I have currently been having some trouble regarding the validation of a contruct which I have generated. I have used the pGEX6p1 vector in which my insert was ligated. The ligation product was further used to transform XL1 blue E. coli cells. The colonies obtained were further used for plasmid extraction as we were finding it difficult to perform a colony PCR. The extracted plasmids were validated for the presence of the insert by PCR and sequencing. Both these methods yielded a positive clone which I wanted to validate finally with restriction digestion.
However, as opposed to the PCR amplification I have obtained 2 insert bands as shown in the figure. I have tried to culture the vector again to detect any possible contamination, however, the same results were obtained.
1. Could someone please provide some suggestions as to why this is happening and whether I should believe the sequencing results as I obtained a clean insert sequence.
2. I also wish to enquire if therer is any preferrede strain for pGEX6p1 vector as I am contnuously obtaining a really low yield for thsi vector?
Desperately need some suggestion..