hello all,
I have transformed a 20kb plasmid into E.coli JM109. But the problem is the growth time has become too high, may be more than 30 hrs and so. Colonies start appearing on plants after 24-30hrs but when i pick them up and inoculate in liquid media containing 50ug/ml Kanamycin, they are not growing. I am sure about the transformation of correct plasmid.
please give some suggestion to speedup the growth.
thanks
How old are your kanamycin plates? The answer might just be that your plates are going bad and you're getting background colonies.
If you're 100% sure they contain the plasmid and aren't just background colonies then maybe try lowering the kanamycin concentration to 25 µg/mL. We use 50 µg/mL for medium to high copy number plasmids but growth is often really poor if we use 50 µg/mL for a single copy kanR insertion in the chromosome, so we use it at 25 µg/mL instead. If your plasmid has a very low copy number maybe that's the issue.
Seems like antibiotic problem or negative result for transformation.
1) Is your plasmid resistant to kanamycin?
2) Try using fresh kanamycin plates ( 50ug/ml)
3) Normally, when i transformed using JM109 with 13kb plasmid, the colonies were visible after just overnight incubation at 37C (16-18 hrs), if tiny colonies appear after 24-30 hrs, those are probably false positive. Colony PCR done on those tiny colonies showed that there was no inserts at all. So, it's safe to assume that if there's no colonies growing after overnight incubation, then it's negative result for transformation.
4) If it's negative result for transformation, try checking if your plasmid is sufficiently linearized, if your inserts have the correct and sufficient homologous arm (if you are using homologous cloning), if your molar ratio of vector:insert is correct
- Are you saying that the problem is both slow growth in Liquid Lb + kan as well as LB agar plates plus Kan ?
- If so this might be something intrinsic to your construct, especially that size interfering with the growth of your host strain
- Apart from attempting to grow under more relaxed consditions s suggeted aabove you could also try re transforming into another host strain where growth might be more permissive
- In addition, you could also try growing in an enriched medium like 2 x TY instead of standard LB and in addition, if the problem is growth in liquid culture as well, you could try allowing your transformant to grow for about 1 hour in 2 x TY before adding selective antibiotic
Thanks to all for suggestions. I am trying one by one and lets c which one works best for me
if u have a problem with kenamycin then u should change it. befoere using autoclave surely all things.
if you are getting growth of colonies after 18 hrs then definitely it is not a transformed one which you are expecting
see if your plasmid is resistant to kanamycin or ampicillin or tetracycline or combination of any two or all three, then once again try by preparing fresh plates
check once again all the transformation procedure done exactly or repeat the experiment with fresh culture and plates to get the good results
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