SFLANAGAN@BIONET-20.BIO.NET (Steven D. Flanagan) (04/14/89)
RAY@leicester.ac.uk SFLANAGAN@bionet-20.bio.net Dear Ray, I am cloning blunt end PCR fragments (216 bp) into Bluescript plasmid using Not1 linkers (ligated after PCR). The yield is very low but I have obtained 12 recombinant clones. One problem that I have encountered with Bluescript is that the color selection tends to disappear with small inserts, especially if the insert is in the reading frame. Perkin-Elmer/Cetus Amplitaq is highly purified and reliable. I haven't heard of exonuclease problems with either the enzyme (J.Bacti. 127(2):1550-7) or other commercial preparations. If you write to me with more specific problems or questions, I may be able to give you protocol and/or references for cloning and screening. Good luck! Sincerely, Heidi Bauer Neurosciences Division Beckman Research Institute of the City of Hope Duarte, California, USA -------