Hi Mark, 0. 1. Please note that you don't HAVE to have NdeI/ClaI cuts in your vector, as both enzymes have several compatible end cuts. It's not as good for NdeI but ClaI has decent numbers of compatible ends.
2. Unless your gene is super-huge or really difficult to PCR, I see no reason to avoid a PCR subcloning step with (very cheap) primers dsigned for more user-friendly digestion. NcoI is a lot friendlier - it has useful compatible ends and it is present in a huge variety of vectors (virtually all pET for example). Most of the time if you use proofreading polymerases (e.g. PfuUltra or KOD) you will not see any errors at all (very rare with amplicons under 3000 bp). Primers to suclone something simple like that should cost ~4-7 dollars each in the US (no idea how much is it in the UK). -----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Mark J. van Raaij Sent: Saturday, July 19, 2008 7:15 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Expression vector with NdeI-ClaI sites Dear Bulletin Boarders, We have a protein cloned NdeI-ClaI in pT7.7, which expresses insoluble. Ideally, we would like to do a simple sub-cloning using NdeI and ClaI into another vector with an N-terminal His-tag, to produce large amounts of insoluble protein which can then be easily purified by metal chromatography in denaturing conditions. We are not looking to crystallise this particular protein, it is for other purposes. I had a look at various vectors (pET, pQE and others), but did not find one which has NdeI (CATATG of which the ATG would code for the first Met of our protein) as a 5'/Nterm cloning site and ClaI (ATCGAT) at the 3'/Cterm, and an N-terminal His-tag. Does anyone know of a commercially or otherwise available expression vector with these characteristics? For good order, if it is commercially available, we will buy it, we are not looking for a free ride here... If finally there is no such vector, we will just order primers and do PCR-cloning, sequencing etc., but I thought I'd try this first, as the vector may be useful for other projects, but the primers won't. Greetings, Mark
