since my insert is unstable, i am using NEB stable supercompetent cells. After transformation I get clones but with no plasmid in it..im using recommended ampicillin (100ug/ml) to make my plates..any suggestions?
Can be a number of things, form the transformation protocol and Plasmid Preparation protocol to DNA extraction and confirmation. Do not think this is enough information to give an answer.
Yes, we have used these bacteria. However, like Jon has mentioned, your problem appears to lay elsewhere. Plasmid -free bacterial clones can't be selected on plates containing antibiotics. Either your bacteria are contaminated or Ampicillin has gone bad. It is also possible that antibiotic was added into hot medium that resulted in antibiotic degradation.
Colonies with no plasmids will suggest that either you are getting some unspecific strain of bacteria growing due to problem with antibiotic or whatever else....To rule out antibiotic issue take untransformed cells as control...if u get colonies on your transformed cells but not on control and still you dont get plasmid after midi prep would suggest some problem while picking the colonies from the plate....do u incubate longer to get bigger colonies...sometimes in such cases antibiotic is exhausted and the bacteria will just try to get rid of the plamid and u might get satelite colonies with no plasmid...Do u check the OD before plasmid prep..............? .just take some clone and do colony PCR .....some people even add 1% glucose to plates and broth to overcome such issue......Other easy way would be try with other cells such as stbl3...who knows the strain is not liking your plasmid.....or try another plasmid with a different selection marker....
thanks
hi all, so
.I plated again and there are hundreds of colones and no colonies in my negative control...however the colonies are growing in two states..in 14 hrs I see big colonies as well as lots of satellite colones all over the plates..i picked the colonies and grew them overnight..they did not grow at all or took really long times (3days). I did miniprep with the cells that grew, but see no plasmid..any suggestions? Why am i losing the plasmid and why am i getting colonies in two stages and why they are not growing in liquid culture..im using the same antibiotic with same concentration for preparing plates and liquid culture..
Does anybody have experience with using NEB stable supercompetent cells? - ResearchGate. Available from: https://www.researchgate.net/post/Does_anybody_have_experience_with_using_NEB_stable_supercompetent_cells [accessed Jan 22, 2016].
Can you attach a photograph of your plate? Unfortunately you are not providing with enough info. Are you dealing with direct repeats? Viral genes? The vector used to transform cells is so-called cloning or expression vector? If it is an expression vector, the gene is expressed under which promoter? It is a high or low copy number vector? At what temp you incubate the plates?
Cultures are growing under selection and plasmid cannot be isolated simply does not make sense. Never experienced or heard of satellite colonies on a plate containing 100 ug/mL ampicillin and that too after 14 h incubation. Bizarre!
hi Syed, thanks a lot for reply..please see the pic..the pic was taken after 24 hrs incubation at 30 degree..left dish:negative control, central: vector only, right: ligation product. to answer your question..i dont know what kind of repeat i have in my gene..i put the seq in the repeat masker it shows repeat and it probably says simple repeat..i dont know what simple repeat means..it is a human gene..the vector in mammalian expression vector with CMV promotor and it is low copy no. vector..i plated in 30 degree Celsius..please help..it is driving me crazy..im trying it so long..
HI Fazlur,
I am having the same issue with NEB stables for sometime now. How did u solve the problem in the end? Curious to know if and how you sort the issue.
Thanks
Hi there
I have come across the same quetion with NEB stable cells. The plasmid concentration is about 50-100ng/ul for mini prep(stripped with 50ul). I am thinking it may be cause by large vector size and broth formular. I am using 2YT for overnight culture and my vector is about 15kb
And I found possible reason for this strain on NEB website:
Recombination Deficient: (recA1) E. coli has a repair system that will recombine homologous sequences. Genomic clones often have duplicated regions, and recA mediated rearrangements can be problematic, particularly when regions of homology are longer than 50 bp. Strains that have the recA function deleted tend to grow more slowly than recA+ strains.
I had the same issue with using Lenticrispr v2 plasmids. Simply called, asked for refund, and went back to one-shot Stbl3 from invitrogen.
Works flawlessly!
We ran into the exact same problem, chased after a none existing plasmids for awhile. Started from scratch and just used old fashioned DH5alpha's and got what we wanted. Just came across this sorry other people were having yrouble glad I'm not crazy
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