If the number of colonies are very high (even hard to count on plate) when transformation done with 1 ng of DNA then how to calculate transformation efficiency? Anyone experienced that even with 1ng DNA transformation plate showing more than 1000 cfu. Please share your experience.
Hello Mangesh,
Usually, 1 ng is too much and will saturate the competent cells. We would use Invitrogen DH5 alpha MAX efficiency cells and aliquot them after the first thaw. When first thawed, the transformation efficiency would be around the advertised 1 x 10^9 CFU per ug of DNA. Those cells are supplied with a pUC19 tube that is at 0.01 ng per ul and I would use 1 ul to test the efficiency, or 1/100th the amount you used. The size of pUC19 is on the low MW end for standard plasmids, so it goes in a little better than larger plasmids but is a good standard. So I would recommend using 0.1 and 0.01 ng of an intact, well purified plasmid DNA to test the efficiency. Otherwise, you will find that as you increase the DNA concentration, the # of CFU will increase but the CFU per ug of DNA will decrease.
Hi there,
1000 transformants for 1ng of DNA corresponds to 106 transformants for 1µg which is actually the lower limit for cell competency in a cloning context. So it's usual to get such results. As suggested above, the use of 0.1ng or even 0.01ng of DNA makes the things easier. And the plating of dilutions of your transformation mix is advised for easy counting.
In fact the "magic" number of 109 cfu/µg is only an extrapolation of the result of transformation made with a fraction of ng and absolutely not the result actually obtained for 1µg of DNA engaged in the transformation mix.
Thank you Christopher S Morello and Dominique Liger for your reply.
Actually I prepare myself chemically competent DH5 alpha (E.coli) cells in lab using cells from Invitrogen. I check the efficiency with 1 ng of DNA. I get transformation efficiency in the range of 1 x 10^7 to 1 x 10^8 CFU/ ug of DNA.
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