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Direct Cloning of Blunt-end PCR Fragments

Direct Cloning of Blunt-end PCR Fragments

BioTechniques 13:613

I run the entire thing out on a 1.3% agarose TAE gel, cut a trough in front of the band, pour in some 0.7% LMP agarose(BRL), run the product into it and excise.

The PCR product in the LMP can be used for ligations directly, without purification. The ligations take place at room temp on the benchtop. I prepare the vector with minimal digestion (~2hr) then treat it with shrimp alkaline phosphatase(USB). I usually prepare a stock of this vector to have on hand, so I know it is good and will have a low background. You may also want to try using an EcoRV cut vector instead of a Sma cut vector.

you can then just "shotgun" clone this DNA. However, if you have several non-specific bands then you may want to gel purify the fragment first.

One point to note....

We have given up blunt end cloning into Sma I sites where possible. Several people have reported problems with Sma I cut DNA. By choice we clone into EcoRV sites.