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.
- Phenol extract the PCR product
- Ethanol precipitate
- Treat for 1hr at 37C with 10 units of T4 DNA polynucleotide kinase and 10 units T4 DNA polymerase I(NEB) in a 100ul reaction volume containing 50mM Tris-HCl pH7.5, 10mM MgCl2, 1mM DTT, 50 ug/ml BSA, 1mM ATP, 200uM each dNTP.
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.
- Remove the oil with Diethylether.
- To 40 ul of the PCR reaction add 50 ul of H2O. Add 10 U T4 PNK, 10 U klenow, ATP (to 1 mM) and some more dNTPs (usually 4 ul of 1.25 mM...whatever) and icubate at 37 oC for 30 min.
- Phenol/chloroform extract
- chloroform/IAA extrac
- EtOH precipitate
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.