I tried to transform the pET32a plasmid in dh5 alpha cells a couple of times. The first time I got no colonies after spreading the bacteria on the ampicillin plate, and the second time I only got 3 single colonies.
I also transformed another plasmid which contained an ampicillin resistance gene in the same dh5 cells, to make sure my cells are competent, and got a plate full of colonies.
Is it normal to get only three single colonies after transformation?
Are these single colonies reliable enough to use for plasmid extraction?
Hi there, With the view of plasmid amplification, one transformant is enough. Are you sure about the amount of plasmid you engage in the transformation? If you get very few clones for one plasmid and loads with the other it might be due to the amount of circular plasmid in the transformation mix. If you want to make sure that the few clones you get are relevant you have to run the control experiment without plasmid to check the behaviour of the recipient strain on selective media.
Dominique Liger Thanks for the answer. I used around 8-10ng of each plasmid, and got a completely empty plate for the no-DNA control.
Hi again, if the control is clean then the clones are OK for plasmid amplification. Have you checked the plasmid integrity on agarose gel? I suspect it is either partially degraded or vastly non linear (ie. not suitable for a successful transformation).
Dominique Liger No, I haven't extracted the plasmid yet. I just made some glycerol stocks and put them in the freezer.
But, I'm sorry I didn't completely understand your comment. I'm planning to do restriction digestion on the pET32 plasmid and use it as a construct for the insertion of my gene of interest. What if I found out the plasmid was vastly non linear or partially degraded after checking the integrity? Would that cause any problem for the downstream application?
My previous comment was applicable to the plasmid sample you have used for transformation. Efficient transformation occurs only when the plasmid is circular. If most of the sample is linear less clones are obtained.
Improve your efficiency by:
- Making sure your plasmid is not degraded or has impurities like proteins or other components.
- Make sure your plasmid is in water.
- Increase the incubation time after you transform the cells, if the incubation was 1 h increase it to 1.5 h.
- Use fresh competent cells. Frozen cells lose efficiency over time.
- Test other mediums. I suppose you used SOC.
- Decrease the concentration of antibiotics.
- Test transform with different concentration of your plasmid
- If you have an electroporator, try electroporation.
Good luck!
Getting few colonies are may be
because of old or improper preparation of competent cells
Wrong concentration or old stock of antibiotic in selection media
Wrong proportion of plasmid and insert
Preferably to make use all the media, plasmid, amplicon, agar, antibiotic freshly and try.
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