pGEM-T Easy vector contamination - (May/07/2012 )
- Hello everyone.
- I want to subcloned a 760 bp insert (Which is cloned in pGEMT vector) into a histag fusion vector pQE30 (3461 bp)
- First I digest pQE30BG (4184 bp) (that has another insert, beta giardin) to have my 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:
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 (less thean 4) and after performing minipreps I see on gel that they are all false positives. I get pGEM-T Easy vector contamination.
pGEMT vector band is around 2500 base pairs, but pQE30 band should be around 3461 bp.
Anyone knows why this contamination happens?
SacI is a pain in the neck ...sensitive to salt concentration ...can be that it did not cut properly and that this is the reason for background resulting from the re-ligation of vector that is just cut with KpnI.
You can try to treat you vector with phosphatase ...and check the purity of your plasmid preperation ...NEB says: "SacI is inhibited by salt concentrations > 10 mM. Mini-prep DNA containing residual salt is resistant to cleavage. A 70% alcohol wash or dialysis can be used to remove the salt."
Be aware of that!
I think why you used the PCR purification kit to purify your products after the 1st enzyme digetion. If the Sac I reaction condition is so particular, why you firstlly digest with Kpn I, purify and then perform the Sac I digest.
In the Takara bio catalog, it prodide that the Kpn I and Sac I could be perform double digestion in the L buffer. If you want to, this may make efficiently for you digetion, a recommend enzymatic systems is, ~ 1 ug plasmid DNA, Buffer and 1 ul for each Sac I and Kpn I and plus 1X BSA for your reaction vol. up to 20ul and 37 celsius for 4hr and run the 1% AGE to make a gel recovery. Usually, you'd better to make a control without insert to see wether your empty vector cut efficienty. If not, the control plate may grow many clones.
Still another problem, how do you identify your miniprep plasmid DNA, digestion or PCR or sequencing? Given you have aminiprep, you cold make a double digest used 3-4 ul plamid in a 10 ul double digestion vol with Sac I /Kpn I, and incubate 1hr to run a 1% AGE. this may be evdent to judge yourpositive ones.
Often if you run a good 1% AGE, it may be clear to judge your cut efficiency according to the Marker,due to the conformation of different types of plasmids.
Anyhow, good luck.