pGEM-T contamination

Miguel Azevedo via methods%40net.bio.net (by macazevedo from gmail.com)
Thu Jul 26 17:18:29 EST 2012


- 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



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