I have tried cloning my GOI in topo TA cloning vector with given protocol provided with the kit. But everytime i get colonies in Kana plates which tend to be false +ve.
The Topo kit worked efficiently for another gene of 2.5Kb size.
Is it because of large size of my gene that it fails to clone in Topo TA?
I heard of another kit of TOPO® XL PCR Cloning Kit for 3-10Kb PCR product.
Should i go for it?
Or should i try pGEM vector? What is the maximum size i can clone in it?
Hello,
6.2 kb is big. I have successfully cloned a 7.kb fragment, 2 500 bp fragments in an expression vector. I used Gibson cloning method for this, may be you could try that too.
Good luck
I would suggest using gibson assembly. PCR amplify fragments of gene and vector with overlapping ends and join them together using homemade recipe or NEB gibson assembly/HiFi kit
thanks Nivedita Awasthi and Shyam Solanki..
Well i have tried USER cloning as well but not showing any colonies. I'll try Gibson assembly this time. Thanks.
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