Genomic cloning troubleshooting

Genomic cloning troubleshooting: Evaluating the success of partial fill-in reactions with the Xho I half-site arms cloning strategy

Gary Kobs
Promega Corporation

Promega's LambdaGEM®-11 and LambdaGEM-12 vectors are designed to take advantage of a cloning strategy (1) that relies on the exclusive specificity with which partially filled-in Xho I arms can be combined with partially filled-in Sau3A I digested genomic DNA. The only ligation products possible are single copies of genomic inserts with appropriate arms, since the partial fill-in prevents self-ligation reactions of vector arms, central stuffer, and genomic fragments. The principal advantages of this method are that it makes genomic DNA fractionation and dephosphorylation unnecessary, is very rapid, and requires small amounts of starting material.

Last update 15-Jun-2006, Rating of 2 votes.

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	By t.srinivasan on 15-Jun-2006 i was used pET28a vector for double digestion using Nhe1and Xho1. in digestion, 1% agarose gel shows smear like bands. finally i got the negative results in cloning, so what is the reason behind this. please suggest me. and one more doubt why supercoiled plasmid first come down, the followed relaxed plasmid. thank u. Rating: Reply

	By t.srinivasan on 15-Jun-2006 what is explanation of pET28a(+) vector. Rating: Reply

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