i am finding difficulty in cloning my genes of interest into pET28a vector. I am using E. coli DH5 alpha cells as cloning vector and BL 21 cells as expression vector. competent cells were prepared by calcium chloride method and ligation was done at 1:1 and 1:3 molar ratio. total DNA concentration used for transformation was 25 microgram per microlitre. What could be the possible reasons for this??
Whenever you prepare competent cells (from any E.coli strain), you should test the competence with a known plasmid. 1 nanogram of pET28 in 100 microlitres of competent cells should give you 500-2000 colonies depending on the strain. DH5 alpha is a slow grower compared to others, it is also slightly less competent, so 500 is probably the best you get.
Apart from that, I cannot advise when I don't know the problem. 1) Do you get no colonies after transformation, or 2) do you get colonies but they are all empty vector? If it is the former, make better competent cells and test them first. If it is the latter, 25 microgram per microlitre for transformation is incredibly high. We usually do our ligations with 50 nanograms of vector and 50-100 nanograms of the small fragment depending on size (if it is very small, we use less than 50 nanograms) in a total volume of 20 microlitres, and we only use 5 microlitres of that for a routine E.coli transformation.
Thank you sir for your advice
initially i was not getting colonies after transformation. recently i got positive clnes after 2 days of incubation. the same bacterial starins and vectors as described before were used.
same problem I was facing 6 months back..in my case, competent cells were working fine but ligation and transformation did not gave me any colonies. I had pCAMBIA1300 vector and my gene of interest were 2 kb. No 1:3 or 3:1 molar ratio gave me desired result. It was just due to the impurities like protein or phenol contamination present in the midi DNA preparation so that also after using Qiagen PCR clean up kit did not helped me. I had done phenol-chloroform step repeatedly to eliminate any protein contamination present and then I had done chloroform isoamylalcohol step 3 times very carefully so that the lower layer doesn't come with the upper aqueous layer. Then like normal procedure I have precipitated the DNA with sodium acetate and absolute ethanol. For ligation, I had taken 200 ng of vector and 100 ng of insert, but still it worked well. The colonies came and the problem was solved.
Clean DNA certainly helps, another very common problem is the ligation buffer, if it is melted and frozen repetitively, the ATP can degrade, and this can be easily solved by aliquoting ligation buffer (20 microlitres aliquots) when you use it the first time (don't forget to mix well after defrosting) so you don't melt and freeze more than once or twice.
Simple to do but it works wonders :)
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