I have 3 synthetic DNA fragments, having different restriction sites. (Fragment1: Spe-I/ Apa-I; Fragment 2: Apa-I/ Eag-I and Fragment 3: Eag-I/ EcoRV cloned into different plasmids. Now I want to clone them in a single vector in one series. Please tell me the easiest way to do that.
The easiest approach would be to do a bulk ligation with all 3 fragments and the use primers terminal to the ends of the desired product to amplify the correct ligation product. Clone the amplified product after appropriate digestion.
Over time I used more and more a PCR-based cloning method (see publication attached for the method) instead of the traditional digestion and ligation. It was working perfectly in my hands several times and there are no limitations with regard to sequence or restriction enzyme. Perhaps it would be also useful in your case. If you decide to try this method but face problems, please feel free to contact me. Good luck!
Article FastCloning: A highly simplified, purification-free, sequenc...
You can do it with bulk ligation - all fragments in one tube, or better and maybe easier use gibson cloning, where for sure you have to built new primers.. :-)
I you want to ligate the three fragments into single vector with high fidelity, you may go for recombination prorocol and can use any good recombination kits which are faster and very reliable.
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