From owner-drosophila@net.bio.net Tue Mar 26 22:00:00 1996 Path: biosci!LAMAR.COLOSTATE.EDU!ashok From: ashok@LAMAR.COLOSTATE.EDU (Ashok Mudgapalli) Newsgroups: bionet.drosophila Subject: Subcloning large PCR fragments for transformations! Date: 27 Mar 1996 14:58:48 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 26 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Dear Netters I have amplified a PCR fragment of about 11.5 kb from D.melanogaster genome using Boehringer Mannheim's Expand Long Template PCR kit. I tried to subclone this fragment in to casper transformation vector and in other vectors with no success. Basically the PCR product has 3'-A over hang and I tried to polish the ends with Klenow or T4 DNA polymerase and tried to do blunt end ligation in to the casper vector. It did not give any positive clone. I also tried Stratagene's pCR script ligation kit. Here I have size limitation, the vector provided by the Stratagene can take fragments upto 10 kb. I tried this vector as a shuttle vector. This procedure also did not work. I have been subcloning various fragments (up to 15 kb) in to the casper vector without any problem, of course they are not PCR products. I would greatly appreciate if some could provide me few hints or their experiences in subcloning large PCR fragments in to the casper vector. All commercially available T-vectors do not have the capacity to hold such a large fragments (that was the information I got from different vendors). Thanks in advance to every netter Dr.Ashok Mudgapalli .