arenas@IAGO.CALTECH.EDU (Jaime Arenas) (09/14/90)
> I'm looking for someone with experience cloning medium sized (500-1000 bp) > PCR fragments into a vector for screening. I'm having troubles getting > transformants to screen. > I discovered through a control experiment that Bluescript vector that I > had cut, blunt-ended with T4 polymerase, phosphatased with CI alkaline > phosphatase, and then kinased with T4 polynucletide kinase would NOT yield > Amp resistant colonies. Unfortunately I don't have the cut, blunted, > unCIAPed vector to determine if the problem lies with blunting or with > kinasing (although either is a problem, since I have to both blunt and kinase > the PCR fragements). Mark, I had similar problems trying to clone PCR products in bluescript. I was trying to blunt-end ligate PCR product into the SmaI site of BS. However, It never worked. Although I never knew for sure what was wrong and to make a long story short, here's what I found and how I did come out of it: 1.- I found that "blunt ending" PCR fragments with T4-pol was not needed indi- cating that there was enough blunt ended molecules in the PCR product. 2.- Always had troubles when cut Bluescript with SmaI. Then changed to EcoRV and worked just fine. 3.- After treatment of EcoRV vector with alkaline phosphatase, I removed it by phenol/chlor (ph 7.0) extraction and three chlor. extractions followed by ethanol precipitation. 4.- After PCR I phenol/chor. and etOH pp. products as above and phosphorylated them with kinase. Then isolated kinased PCR products by running Nu Sieve LMP agarose gel (SeaKem) and cut band out. 5.- Blunt end ligation works just fine adding an aliquote of the melted agarose slice up to 0.3% agarose in the ligation reaction. For this, melt agarose, mix all reagents except enzyme in another tube and heat to 50-55 celcius. Mix melted agarose and pre-warmed reagents. Put at 37 degrees and add enzyme. Leave at 37 degrees 10-15 minutes and then sit at RT for 3 hours or more. 6.- Although this was enough for me. For higher yields purify PCR products away from agarose P.S. If you are using Blue/white selection, never mind the color of the colonies when using Bluescript I. Good luck, --------------------------------------------------------------------------- Jaime Arenas, Ph.D. There is no places, Division of Biology 147-75 there is beginnings and ends, CALTECH but nothing is ever forgotten. Pasadena, CA 91125 ARENAS@CITIAGO.BITNET ARENAS@IAGO.CALTECH.EDU (NO, there are no misspellings)