kanamycin resistance problems.

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oppox
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kanamycin resistance problems.

Post by oppox » Tue Jul 11, 2006 1:13 pm

Im trying to get a pET28 vector into a cell (any cell, ive tried most of them)
the vector has Kan resistance. The problem though is that the cells without vector grow pretty good on Agar-Kan plates, so I cant destinguish between positive cells.
Ive tried to screen with different concentrations of kan (30,40,50,60 and so on), The cells stop to grow around 60.
Ive tried different batches of kanamycin too.
The cells ive used are, xl1-blue, bl21, and alpha something.

Have anyone else experienced any similar problems with Kan.

thx.

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canalon
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Post by canalon » Tue Jul 11, 2006 2:05 pm

Concentration should be OK. But was your solution(s) kept at 4ºC? Sigma do not recommend freezing. xl1 blue, bl21 and DH5α should be sensitive to 50µg/ml kan.
I ususally do not have problems with kan, unlike ampicillin.
Patrick

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oppox
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Post by oppox » Tue Jul 11, 2006 4:26 pm

So not freezing the kan solution? ok.
In novagens manual, the working concentration of Kan is 30.
Your cells do not grow untransformed?
thx for the help.

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canalon
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Post by canalon » Tue Jul 11, 2006 9:43 pm

No, at least not JM109, and IIRC DH5α, although I discovered only very recently about the freezing of Kan, and I did not had problems either.
Patrick

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oppox
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Post by oppox » Tue Jul 11, 2006 9:55 pm

hmm its something funky about this, I made fresh never frozen Kanamycin today and I did a new conc test with untransformed bl21 cells. Hope I can see some results tomorrow, thx for the help.

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Post by seaseasea » Wed Jul 12, 2006 6:28 am

hello,
freezed kana could be no problem.i usually use freezed kan.
phenomenon like this is really rare.
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Post by mkwaje » Sun Jul 16, 2006 9:42 am

Yes, at our classes we usually freeze kan for storage; during the first semesters of teaching mol bio, there were no problems with kan resistance but after several years of using the same cell stocks, I noticed kan resistance developing. I am careful about contamination, but it seems the cells have been developing kan resistance after a while. I was at a loss explaining this so I simply resorted to using a new vector. Resistance of the cells reached up to 60 ug/ml.

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Post by qq-wheat » Wed Aug 16, 2006 2:36 am

Yes ,we usually store kna at 4 degree .
cheers

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