(by cathalgarvey from gmail.com)
Fri Jul 27 04:08:22 EST 2012
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
> (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
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> Methods from net.bio.net
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