indeed a good trick if compatible with your strategy, in other cases, simply using high-fidelity variants of enzymes helps to minimize star activity artefacts by orders of magnitude (according to published reports for some NEB enzymes) best wishes/merry x-mas and all the best for the new year, David
2009/12/23 DK <[email protected]>: > In article <[email protected]>, "Jayakumar, R" > <[email protected]> wrote: >> >>You mean you want to linearise the construct by cutting it with a RE >>outside the expression cassette before transformation??? Is that what >>you mean. I don't see the purpose of cutting a vector after ligating vector >>and fragment to clone a PCR fragment efficiently. It does not make much >>sense. > > It does make sense! Sometimes. In special or very hard cases. > The original is hard to interpret so I won't comment. But with regard to > your reply, I can offer two cases where cutting ligation mix makes > sense: > > Say, MCS contains sites ABCDEF and you clone your insert > by A and E. If, for whatever reason, your ligation worked very > poorly (but ligase did work), you will mostly get back your original > plasmid. However, if your insert is not cut with enzymes B and D, > you could have cut the ligation mix with either B or D (or both). > Since linear DNA is 1,000-10,000X less transforming, if you get > any clones after doing this, almost all of them will be positive. > > Even if you are doing everything 100% right and you ligations > work wonderfully, there are several circumstance where doing > the above makes sense. I had the following cases: > > 1. Huge plasmid that needed to be cut with an enzyme that cut > it incredibly poorly (and also had start activity). Because of the > size, there was no easy way to separate cut and uncut forms. > But couple of other sites that would be excised in the positive > clones cut it without any problems. > > 2. Blunt cloning that destroys site for the blunt cutter. (In fact, > with a little foresight, this can be used to do directional > blunt cloning). > > 3. Quick and dirty protein expression tests when ligations > work/should work very well. Transform cut ligation mix > directly into expression strain, don't select clones but scrape > everything off the plate (which should be >>99.9% positives) > and use it as a starter culture. This way, the day after ligation > one can induce and run an expression gel to, say, check > expression level/solubility pattern of the new construct(s). > > DK > > >>-----Original Message----- >>From: [email protected] [mailto:[email protected]= >>=2Eindiana.edu] On Behalf Of Nasser Mahna >>Sent: Tuesday, December 22, 2009 12:23 PM >>To: [email protected] >>Subject: Cloning trick >> >>Dear All, >>I would to ask if there is anybody mastered in working with restriction a= >>nd digestion. >>I want to clone a pcr fragment inside a binary vector. To do it efficient= >>ly, I want to use a restriction enzyme after ligating vector and fragment= >>, which cuts the original uncut vector before transformation. >>what method do you offer? >>Best regards, >>Nasser. >> >>_______________________________________________ >>Methods mailing list >>[email protected] >>http://www.bio.net/biomail/listinfo/methods >> >> >>This email message may contain legally privileged and/or confidential inf= >>ormation. If you are not the intended recipient(s), or the employee or a= >>gent responsible for the delivery of this message to the intended recipie= >>nt(s), you are hereby notified that any disclosure, copying, distribution= >>, or use of this email message is prohibited. If you have received this = >>message in error, please notify the sender immediately by e-mail and dele= >>te this email message from your computer. Thank you. >>--_002_AE06C4610E75404183F550518E585DBC264B821D17MSXMBCCR2rosw_-- >> > _______________________________________________ > Methods mailing list > [email protected] > http://www.bio.net/biomail/listinfo/methods > -- David Minde MSc (TUM) Cellular Protein Chemistry Room 707 Department of Chemistry Faculty of Science Utrecht University Krytgebouw Padualaan 8 NL-3584 CH Utrecht The Netherlands office phone +31 30 253 4105 (magic:) android +31 652614443 Science is what happens while we are making other plans (~John Lennon) _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
