Hi !
Has anyone tried multiple fragment cloning for bigger PCR fragments ? I need to clone a 3kb,3kb and 4kb PCR(total 10kb) fragments into GFP vector. I have difficulties as the fragment size is little big. Can anyone please help or suggest. I would appreciate if anyone who has done this share their reaction conditions. Or can anyone suggest which is the best method. Thanks in advance. :)
In-Fusion cloning (Clontech) / GeneArt® Seamless PLUS Cloning and Assembly Kit (Life technologies) can help you.
Hi Kamal Kumar !! Thank you for the suggestion. I tried with this system as well. But the problem is bigger fragment sizes.
Do your fragments have overlapping ends? if so, you could try cloning by homologous recombination in S. cerevisiae. That way I successfully got a 7kb (in 3 fragments) gene into a vector.
Do Gibson aseembly to have all fragments in one vector. You just have to design the right overhangs from each fragment you want to assemble. NEB is selling a ready to use mix. To understand it better please click the link below
https://www.neb.com/products/e5510-gibson-assembly-cloning-kit
I agree with Christian. If you are trying to clone multiple fragments in tandem, Gibson cloning will be superior to sticky-end mediated ligation. The NEB mix does work extremely well. If instead, you are simply cloning 3 different cDNAs to generate 3 different expression constructs, you should be able proceed via standard ligation conditions, irrespective of the fact that your DNA inserts are 3 kb or larger. Just make sure your total DNA concentration in the ligation reaction is less than 100 ng, and try a 1:2 or 1:1 ratio, rather than 3:1 of insert:vector.
Do all these fragements of 3kb,3kb and 4kb PCR(total 10kb) represent the single gene? If yes and you want to expreess them as a single reading frame, you can add them by starting with the AUG codon at the beginnig and continue with out any stop codon inbetween. But cloning 10 kb fragment will be difficult task anyway you try to clone and express.
all the best
did you check Addgene to see if all or part of the plasmid has already been constructed? Addgene has 40,000 plasmids...there is often something there that can help..especially fluorescent protein vectors.
Would you like to share on your vector's selection? Are u using the GFP vector and what makes u decide on GFP vector? Thank you!..
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