ligation problem - (Aug/04/2005 )

i want to ligate 6-10 kb mixture of fragments digested with genomic dna in pbluescript vector ( stratagene) but phasing a lot of problems, several time ligation reaction failed. vector and insert both are eco r1 digested. and quiagen column eluted. please suggest me something to overcome problem

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Hi praveen,
I would consider using a different vector backbone such as pGEM11. I have successfully cloned 25kb of genomic DNA into this vector. Some vectors just have difficulties with large inserts. I would also make sure that your bacteria can handle large constructs.

Cloning and stable maintenance of DNA fragmentsover 300 kb in Escherichia coli with conventional plasmid-based vectors
Quanzhou Tao and Hong-Bin Zhang

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thanks for reply
can i know about company which providing this vector, is their blue- white selection with this. please reply soon.

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Promega. This plasmid does not have lac Z blue-white screening.

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