From owner-drosophila@net.bio.net Thu Oct 19 23:00:00 1995 Path: biosci!LAMAR.COLOSTATE.EDU!ashok From: ashok@LAMAR.COLOSTATE.EDU (Ashok Mudgapalli) Newsgroups: bionet.drosophila Subject: What E.coli strain should I use? Date: 19 Oct 1995 19:30:10 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 27 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Dear Netters I am trying to subclone some DNA fragments (12-20 Kb) in Drosophila germ line transformation vector, CaSpeR. The size of this vector is 7855 bp with white gene in it. I am having hard time in getting the ligated plasmid intact after transforming the bacteria. I used ligation kits from Boeringer M, 5prime-3prime, NewEngland Biolabs. All the kits are working well because I could subclone about 7 DNA fragments (3-9 Kb) in blue script without any problem. I used either DH5 alpha or XL1-Blue cells for transformation. These both strains are working with blue script but not with CaSpeR. Since the CaSpeR has invert repeat sequences, I suspect there is lot of rearrangement and restriction going on in the bacteria. Though these bacteria are rec-, I do not understand why I am getting the DNA fragments in between the sizes of vector and insert (after restriction digestion). I am losing major portion of the ligated construct after transformation. I am preparing the plasmid from transformants using mini-boiling prep method. I know lot of labs are using CaSpeR as a transformation vector with their DNA fragment in it. I would greatly appreciate if some body could give me the details of their ligation protocol and the strains of the E.Coli used for successful transformation. Many thanks to every body in advance Sincerely Dr.Ashok Mudgapalli .