pGBKT7 miniprep problem - (Jun/04/2005 )
Does anybody have any clue on the miniprep of pGBKT7. It is a Kanamycin resistent plasmid. I transformed the DH-alpha5 with this plasmid and plated the transformants on LB/Kan plate (concentration: 50 microgram per ml), but when i try to miniprep, it just never works. Basically I did not get any plasmid through miniprep, there is no band shown on the gel.
does any body know what the problem is? Thank you very much!
P. S. I use kanamycin monosulfate to prepare the plates, Doea it matter?
nobody knows this plasmid? Does anyone work on the plasmid with kanamycin resistent? can you please give me some clues?
Hi gchambers,
I've been working with this plasmid for about a month now, and I'm having issues with it too. I have been able to get it to grow selectively in Kan25 and I do get colonies out with plasmids. However, my problem is that they never have my insert - I'm trying to ligate in the insert using 2 different cohesive ends.
I don't think it should matter what kind of Kan you use, but honestly I have no idea.
Thanks a lot, AlexG!
I finally figured out that it is because of the concentration of kan in the plate. If the [Kan] is too low, the E coli cells without plasmid can still survive on this plate and even grow much faster than the transformants.
by the way, in your case, I believe by using 2 different restriction sites, the ligation will be a piece of cake.
I've been working with this plasmid for about a month now, and I'm having issues with it too. I have been able to get it to grow selectively in Kan25 and I do get colonies out with plasmids. However, my problem is that they never have my insert - I'm trying to ligate in the insert using 2 different cohesive ends.
I don't think it should matter what kind of Kan you use, but honestly I have no idea.
I've had problems that seem similar to what you describe. I had no problems in growing the vector on LB+Kan (50micrograms/ml) but I did have problems cloning... That was due to the fact that the plasmid was not completely cut with both restriction enzymes. So what I did was cut with one of them, clean the band out of gel and than cut with the other. After that, I cleaned the cutoffs with Microcons (Microcon centrifugal filter devices - Millipore). On my insert I only applied a double digestion, as both enzymes selected needed the same buffer and consequently I cleaned it with Microcons. I noticed also that the restriction reaction is more efficient when performed in larger volumes (e.g. 100 microl instead of 50).
Thanks a lot!
I've been working with this plasmid for about a month now, and I'm having issues with it too. I have been able to get it to grow selectively in Kan25 and I do get colonies out with plasmids. However, my problem is that they never have my insert - I'm trying to ligate in the insert using 2 different cohesive ends.
I don't think it should matter what kind of Kan you use, but honestly I have no idea.
I've had problems that seem similar to what you describe. I had no problems in growing the vector on LB+Kan (50micrograms/ml) but I did have problems cloning... That was due to the fact that the plasmid was not completely cut with both restriction enzymes. So what I did was cut with one of them, clean the band out of gel and than cut with the other. After that, I cleaned the cutoffs with Microcons (Microcon centrifugal filter devices - Millipore). On my insert I only applied a double digestion, as both enzymes selected needed the same buffer and consequently I cleaned it with Microcons. I noticed also that the restriction reaction is more efficient when performed in larger volumes (e.g. 100 microl instead of 50).