Hi Miguel, One way to eliminate background plasmid is to use PCR to amplify the desired sequence, and then perform a DpnI restriction reaction on the product. As DpnI only cleaves Dam methylated DNA, it won't cleave your PCR product but should eliminate the original plasmid.
On 26/07/12 23:18, Miguel Azevedo wrote: > - Hello everyone. > - I want to subcloned a 760 bp insert (Which is cloned in pGEM-T > vector from Promega) into a histag fusion vector pQE30 from Quiagen > (3461 bp) > - First I digest pQE30BG (4184 bp) (pQE30 vector with beta giardin on > it) to have my empty vector (pQE30). > - In order to do that I perform sequential digests with kpnI > (fermentas) in his optmial buffer (10X Buffer KpnI), ethanol > precipitation protocol and then SacI (fermentas) digestion in optmial > buffer > (10X Buffer Ecl136II, PacI, SacI).Do the same procedure for the insert. > - After that I cut insert and vector and I purify with gel Extraction > Quick (quiagen). Then I do an agarose gel to confirm > that I have isolated only insert and vector . Everything is ok. > - The next step I do is ligation (1 hour room temperature and 1 hour > 4º Celsius). The ratio I use for ligation is 1 vector:3 insert > - After that I perfomed transformation as follows with M15 competent cells: > 1) Remove cells from -70°C and let thaw on wet ice. > 2) Gently mix cells by lightly flicking tube. Aliquot ~50-100µl of > cells into chilled, ependorf tube > 3) Add DNA 5 µl to cell suspension and gently swirl tube(s) for a few > seconds to mix. > 4) Incubate on ice for 30 minutes. > 5) Place tube(s) in 42°C water bath for 90 seconds without shaking. > 6) Replace tube(s) on ice for ~2 minutes. > 7) Dilute transformation reaction(s) to 1ml by addition of 900-950µl LB > medium. > 8) Shake tube 200 rpm for 60 minutes at 37°C. > 9) Plate by spreading 5-200µl of cell transformation mixture on LB > agar ampiclin plates containing appropriate antibiotic and incubate > overnight at 37°C. > Next day I get few colonies and all of them are pGEM-T. No pQE30. > I tried to change competent cells (DH5 alfa, XLI blue) and I always > get pGEM contamination. > > May the insert be toxic to E.coli? > Does anyone know why this pGEM-T contamination happens and how to solve it? > > Thanks in advance > > Michael > > _______________________________________________ > Methods mailing list > [email protected] > http://www.bio.net/biomail/listinfo/methods > -- www.indiebiotech.com twitter.com/onetruecathal joindiaspora.com/u/cathalgarvey PGP Public Key: http://bit.ly/CathalGKey _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
