From owner-dros@hgmp.mrc.ac.uk Mon Apr 7 03:12:27 2003 Return-Path: X-Original-To: dros-outgoing Received: from localhost (localhost [127.0.0.1]) by mercury.hgmp.mrc.ac.uk (Postfix) with SMTP id 421AA7D0E4 for ; Mon, 7 Apr 2003 03:12:27 +0100 (BST) X-Original-To: dros-list@hgmp.mrc.ac.uk Received: from localhost (localhost [127.0.0.1]) by mercury.hgmp.mrc.ac.uk (Postfix) with ESMTP id CFA047D193 for ; Mon, 7 Apr 2003 03:12:26 +0100 (BST) Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 6020) id 088687D0E4; Mon, 7 Apr 2003 03:12:26 +0100 (BST) X-Original-To: bionet-drosophila@net.bio.net Received: from localhost (localhost [127.0.0.1]) by mercury.hgmp.mrc.ac.uk (Postfix) with ESMTP id D999B7D143 for ; Mon, 7 Apr 2003 02:55:51 +0100 (BST) Received: from newsread1.news.pas.earthlink.net (newsread1.news.pas.earthlink.net [207.217.77.51]) by mercury.hgmp.mrc.ac.uk (Postfix) with ESMTP id B825F7D0E4 for ; Mon, 7 Apr 2003 02:55:40 +0100 (BST) Received: (from news@localhost) by newsread1.news.pas.earthlink.net (8.8.8+Sun/8.8.8) id SAA18914; Sun, 6 Apr 2003 18:53:38 -0700 (PDT) To: bionet-drosophila@net.bio.net From: Chris Jones <6kuritaro@earthlink.net> Newsgroups: bionet.drosophila Subject: pCaSpeR-hs cloning problem User-Agent: MT-NewsWatcher/3.1 (PPC) Message-ID: <6kuritaro-B3243E.21555706042003@nnrp01.earthlink.net> X-Complaints-To: abuse@earthlink.net X-Trace: newsread1.prod.itd.earthlink.net 1049680418 65.59.71.228 (Sun, 06 Apr 2003 18:53:38 PDT) NNTP-Posting-Date: Sun, 06 Apr 2003 18:53:38 PDT Organization: EarthLink Inc. -- http://www.EarthLink.net Date: Mon, 7 Apr 2003 03:12:26 +0100 (BST) Sender: owner-dros@hgmp.mrc.ac.uk Precedence: bulk Hi there -- I have a student trying to subclone a 4.1-kb Xba fragment into pCaSpeR-hs. The initial subcloning, from a genomic BAC into NEB's pLITMUS went without a hitch, but she's not been able to transfer the insert into pCaSpeR-hs, which is about 8.8 kb. Cut both with Xba, gel-purify, dephosphorylate the vector*, ligate, transform competent JM109. Analyze plasmids from transformants by cutting with Xba and we see two constructs: a single 8.8 kb fragment which is presumably recircularized vector a 4.1 kb fragment plus a roughly 2.7 kb fragment -- I'm guessing the 4.1 is our insert (though I haven't tested that yet), but what the heck is up with the vector? (* we get the same two plasmids back if we don't dephosphorylate the vector, so that doesn't seem to be the problem.) We've done this three times now, always with the same results. I'm going to spend a little time trying to troubleshoot, but I'm hoping someone reading the newsgroup has heard of this, or better still has had some experience of it. Thanks for any suggestions, Chris Jones -- To reply by email, please remove the initial digit from my address. Make the world a better place: shoot a spammer today! .