problem with competent cells

Bari Zahedi via methods%40net.bio.net (by bzahedi from bccrc.ca)
Mon Oct 5 00:32:55 EST 2009


About a year ago,  my lab and another was getting contamination. We had someone look at what was growing in our cultures and they suggested it was yeast.  We narrowed down the source to our 0.25micron columns that we were using to filter our media and reagents. I brought this up with the company and they did alot of testing and decided that it was not yeast and thus did not believe me that what ever it was was coming from their columns. We stopped ordering columns from them and have never had the problem since. 
I thought i would pass that along. But comtamination could be coming from anywhere really. Here are somethings to try to narrow it down incase you have not done so already. 

When you say contamination do you mean fungus growing on your plates or another plasmid is coming out of your plasmid preps?

If it is something like fungus, then my suggestion is to 
1)go to another lab and borrow reagents from them to grow up your DH5a. 
2) If it is coming from your reagents, then you will have to figure it out through elimination. My suggestion is to try test a bunch at one time- this kinda saves time. You can  also try testing your reagents with  mock transformation with no cells. It may be informative. And perhaps if none of this works see if it is the columns!

 If the contamination is a plasmid, 

3)then streak a bit of your cells on your agar + antibiotic plates and see if anything grows. If it does, plasmid prep it and see if it is the same as the contamination you have been getting.

4) Wash your pipettes well, inside and out and use filtered tips. I have been able to eliminate contamination in this way from sets of pipettes in our lab.  

These are just a few to try. good luck.

________________________________________
From: methods-bounces from oat.bio.indiana.edu [methods-bounces from oat.bio.indiana.edu] On Behalf Of ms [monikagicts1 from gmail.com]
Sent: October 3, 2009 4:29 AM
To: methods from magpie.bio.indiana.edu
Subject: problem with competent cells

hi all,

i want to share one problem regarding DH5 alpha ultra com cells
preparation using inoue method. i am getting contamination as i am not
sure whether it is contamination exactly i donot know but i am getting
good efficiency. everything i am doing in sterile environment. all
buffers i prepared in hood and filtered through .22 micron filter but
i donot know what is the problem. so,  please suggest me what is the
problem.

ms

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