is there any problem with the GV3101 competent cells of mine. I made stock for Agro GV3101 and check its sensitivity and resistance to certain antibiotics. they are growing just in overnight incubation at 28C.
28℃ is the incubation centigrade of the GC3101. it is most siutable centigrade for GV,if the centigrade is higher than 28, the plasmid will be lost.see the instruction book will help you.
How did you grow the bacteria? When you grow the bacteria on plate such that you should obtain single colonies you would need at least 2 days. When you streak out a colony or from -80C vial, you could see growth after O/N incubation where bacterial amount was highest. In liquid culture with small volume O/N incubation is enough to obtain good growth.
Pratima, If your GV3101 is growing overnight, pls check for the contamination. Generally it takes 24-36 hrs if without plasmid. Your r growing in Plate or Suspension....?
I agree, if using liquid media AND inoculum from -80 storage, GV3101 can grow o/n without any problem at 28 C. I add all antibiotics for selection of bacteria and plasmid (usually rifampicin AND gentamicin at 50 ng/ml final concentration, plus antibiotic for plasmid selection), and regularly get o/n growth.
I streaked the cells from stock vial to the plate containing rifamycin. i took 2 days to grow. then i selected single colony to make more stocks for competent cells and grow on 5ml LM with 10ug/ml rifamycin for about 20-24hrs until culture is turbid and then transfer its 2ml to the 50 ml of LM with rifamycin then harvest the cells and then resuspend in 20mM cold CaCl2 and direclty put 100ul prepared competent cells in liq N2 and then store in -80. then to check the transformation efficiency and the sensitivity and resistance of GV3101. i grown them on Kanamycin and rifamycin plate. at that time my competent cells were growing just in overnight on rifamycin but as they are sensitive to kanamycin they are not growing in that plate. transformation also seems fine, i got colonies. I am only worried why they are growing on rifamycin plate in overnight while stock GV3101 took 2 days..
thanks all for your kind replies
thanks
regards,
pr
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