I am inserting a sequence to pET29 and after this ligation I will need to fuse my gene of interest to the same vector.
I was wondering if I need to transform e.coli ( strain Bl21) and produce more DNA before my second ligation? Or I can follow the next digestion- ligation after finishing the first one.?
thanks for your advise.
Hi there,
The product of the first ligation will not be homogenous and not abundant enough to work efficiently with (in ligation mix it's usually about 100ng of total DNA). Transformation/amplification in the right strain (DH5 or TOP10 for instance) will allow you to select the right construct from an isolated clone and to start the second step with pure, rich and homogenous material (miniprep allows to recover tens of µg of plasmid).
Hi Mahbobeh, you'll need to transform E.coli with your ligation products, pick some colonies and grow them in small liquid cultures, then miniprep these and get them sequenced to choose one which contains your correct pET29 ligated plasmid. Do this first and then when you've got the correct plasmid you want, you should do the second restriction digestion and ligate in your gene of interest.
If you don't do them separately like this, you'll have a mixture of ligation products and very little DNA to use in your second ligation, so your cloning efficiency won't be very good. In vector restriction digestions I use 1 µg of DNA, which is quite a lot but I find it works well for me and it means I get a decent concentration of cut vector when I purify it from an agarose gel prior to ligating in my gene of interest - that's why I say your 100 ng ligation reaction would not give you enough plasmid to then carry on with a second cloning experiment. Like Dominique and Mangal have both said, I agree that you should use E.coli which cannot recombine any vector/plasmid DNA - I use the DH5a strain. BL21 is NOT a good choice and I would suggest that you only use it for protein expression experiments, not for general amplification of vectors/plasmids.
Hope this explanation helps - best of luck with your cloning!
Thank you all for your complete and very helpful suggestions. I really appreciate that.
You have to multiply your 1st vector construct before go for the 2nd digestion. Otherwise, it will be inefficient.
First of all, you must do the transformation in cloning hosts like E. coli XL1-Blue or DH5alpha so that you would get a high yield of DNA. Transformation into expression hosts as you are planning would result in loss of your plasmid and you won't be able to extract the plasmid for next processes.
After ligation reaction, transform the ligation mixture in cloning host, select positive clones by using colony PCR and innoculate them in fresh LB media overnight. Then you can use plasmid extraction miniprep kit commercially available to extract the plasmid in large quantities for further processes.
So far, others have already said it all, more precisely @Vikas. Besides, a transformation is significant after any cloning/ligation process and remain the prerequisite for determining its success/efficiency.
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