I have newly transformed agrobacterium with gene of interest. Plated on to agar plate with Kan+Gent antibiotics (right concentartion) my agrobacteria are Gentamycin resistant and transformed plasmid carry Kanamycin, grown colonies were screen for gene of interest. Positive colonies were inoculated to fresh LB media containing same concentration of antibiotics. On first attempt cultures are grown well. I did glycerol stock from grown culture with (1:1, glycerol: culture) and stored at -80 and reinnoculate on next day or few days later no growth has been observed.
Does any one tackled such problem in their experiment? your suggestions and possible solutions are highly appreciated.
Thanking you
Did you inoculate the liquid medium directly from the stock? If so, try streaking the bacteria first on an agar plate and then inoculate your LB medium from the plate.
Dear Stefan Werner,
thank you for your suggestion. I did streak plate from glycerol stock. Cultures were grown on plate. In first attempt they also grew when I innoculated on LB broth. but when I rennoculated from grown cultures to fresh LB broth their is no sign of growth.
I also prepared fresh medium with appropriate antibiotics to find out is there any problem with medium. I have also innoculated different glycerol stock available in our lab and neighbours lab. Some cultures grew and some didnt.
I also asked my other colleagues in department to setup cultures for me from glycerol stock and streaked plates using media prepared in their lab. but again some cultures grew and some didnt.
I enjoyed setting up O/N cultures over whole weekend :). Nothing makes sense for me today. I would be greatful for your further suggestion. right at moment I am running out of ideas and hard to find the mistakes.
Once again thank you for your valuable suggestions
Hi Somanath,
My suggestion, inoculate 100 ul from glycerol stock in to the LB (Without antibiotic), in this case if a single viable colony (irrespective of transferred or non-transferred) exist in stock will grow. Once you achieve growth in broth then plate 200 ul broth on each antibiotic plates (one on Kan, one on Gen, one on Kan + Gen), this way you will come to know for transform or WT is exiting in growth.
If it dosen't work and your all antibiotic stock are fine with proper control growth than one possibility: Is that the plasmid may get cured, but it is rare phenomena when you contiguously grow in without antibiotic medium, in that case your cell loose plasmid under non-stress (wihtout antibiotic) condition
Hope this helps you.
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Materials Science
- Plating