small transformant colonies - (Dec/03/2008 )
Hi every one,
I am tring to clone in pGBKT7 vector, a 105 bp insert and and transform it into E.coli. Initially it was troublesome to construct the plasmid with this insert at EcoR1 and BamH1 sites. I did sequential digestion for the recipient vector(pGBKT7) and droped out the insert by double digestion from pcr2.1 cloning vector. I tried different vector insert ratios for the ligation and did controls with vector alone digested with the above said enzymes+ligase, a control with pGBKT7 cut with BamH1 alone + ligase, a minus ligase control as well. I found no colonies in self ligation contro, approximately same no. of colonies in pGBKT7 cut with BamH1 as that of vector uncut transformed to ecoli(+ve control). All these showed colonies of resonable size. But the plates with vector + insert showed very smaller colonies with no colonies in negative control. The selection is Kanamycin 50 mg/ml. The colonies are not showing the insert in colony pcr as well.
I wonder wether there will be problem in the selection media or the cells dont like the insert for some reason................
It would be great if some one solve this mystery. Thank you!
I am tring to clone in pGBKT7 vector, a 105 bp insert and and transform it into E.coli. Initially it was troublesome to construct the plasmid with this insert at EcoR1 and BamH1 sites. I did sequential digestion for the recipient vector(pGBKT7) and droped out the insert by double digestion from pcr2.1 cloning vector. I tried different vector insert ratios for the ligation and did controls with vector alone digested with the above said enzymes+ligase, a control with pGBKT7 cut with BamH1 alone + ligase, a minus ligase control as well. I found no colonies in self ligation contro, approximately same no. of colonies in pGBKT7 cut with BamH1 as that of vector uncut transformed to ecoli(+ve control). All these showed colonies of resonable size. But the plates with vector + insert showed very smaller colonies with no colonies in negative control. The selection is Kanamycin 50 mg/ml. The colonies are not showing the insert in colony pcr as well.
I wonder wether there will be problem in the selection media or the cells dont like the insert for some reason................
It would be great if some one solve this mystery. Thank you!
I would try both lower (25) and higher (100) Kan concentrations. Getting there would be more important than solving the mystery. Who knows you may solve it in the process!