Can someone help in regards to a problem with low efficiency of competent cell preparation?
I am using the CaCl2 method for competent cell preparation but recently I am facing low efficiency of transformation in new competent cells despite several tries. I always test them by doing transformation of approximately 1 ug of plasmid DNA that used to fill the plate but now I am only observing approximately 20 to 30 colonies each time!
CaCl transformation is always variable as you're working with a biological system. It could be any number of factors so you need to troubleshoot. Look at what you've changed recently. It could be the a new batch of media that you are using or the CaCl. Also, don't discount that it's your DNA that’s off and not your cells. If using a new plasmid prep it may have more salt in it which will affect it or too many freeze-thaw cycles. I'd start by preparing a new plasmid prep and changing your media used to grow the cells and the media used for the out-growth. Good luck!
While I agree the RuCl2 method is a bit better, you should still get excellent results from CaCl2 method, we use it often in my lab. The reasons I have seen that gives low transformation are: 1) strains (some E.coli strains just don't transform well); 2) cells harvested at the wrong density. It is important not to let the cultures overgrow; 3) cells not maintained on ice after CaCl2 treatment. Throughout the preparation of competent it is critical that the cells remain chilled, this includes using a refrigerated centrifuge; 4) harsh treatment, if you vortex the CaCl2 cells you kill many of them. Resuspend and mix gently.
Lithium Chloride method is the easiest, fastest and most efficient for transformation of plasmids less than 6kb. You just need 10 ml culture for 10 eppendorfs of comp cellls and requires only one centrifugation. Try out once.
Thank you all. But I think the problem originate from the plasmid either, because its length is approximately 7kb which decreases the efficiency of transformation. Do you have any other suggestion for this particular problem?
a 7kb plasmid is not that large that it should be a problem. The efficiency will be a bit reduced but not dramatically so. Are you really using 1ug of plasmid? That is vast excess and might even be inhibitory, about 5ng is saturating in a typical transformation.
It might be somewhat lower but you should still get high efficiency. If your plasmid DNA is good and the cells are good then you should still get thousands to tens of thousands colonies if not more. What happens when you use a different plasmid as your control, that will tell you whether it is the cells or the plasmid.
Dr. Yashwant Singh Parmar University of Horticulture and Forestry
Sir why is so that when i am transforming with a confirmed plasmid i m getting pretty much colonies after transformation, but when i m using the ligated product i m not at all getting the colonies. Is it due to the ligation problem or what?
I am having the same problem, i transformed my ligated plasmid to competent cells, but not getting my colonies at all, my competent cells are not very competitive i would like to have some idea of optimizing the transformation with lower competency cells...thanks a lot....
The problem is that ligation is itself quite inefficient for the most part, unless you really work hard to optimize all the conditions. Frequently you might only see a few hundred colonies after transformation of ligated DNA, and that is using highly competent cells. If your cells have 10-100 fold reduced efficiency then you might see almost no colonies. A lot of people make the mistake that when they do a control transformation with pure plasmid DNA, they see a few hundred colonies on the plate and think all is fine. However with pure uncut plasmid you should see thousands or ten's of thousands of colonies, the plate should be nearly covered with colonies.