bacteria cross-contamination due to aerosol during pipetting (View forum version)



donny

Posted 01 December 2011 - 07:31 PM

I've been screening mutant libraries and the libraries keep ending up with the same mutant. I made new libraries and the same thing happened again. I suspect 2 sources : reused electroporation cuvette and aerosol during pipetting. I can easily use new cuvettes but I seriously doubt it is the main cause because I've tried transforming cells in the old cuvettes just to test the sterility and got no colony. Moreover, I exposed the cuvettes to UV at 300nm over the illuminator for DNA gel for 10min before using them. I guess that should damage whatever residual DNA that could have transformed. I'm thinking if aerosol generated by pipetting during cell preparation could have caused the cross-contamination. However, instinctively, I find it hard to believe the (invisible) aerosol could cause such extensive contamination. Has anyone experienced cross-contamination due to aerosol generated by pipetting?

bob1

Posted 02 December 2011 - 02:03 PM

Yes! Plasmid DNA is very very abundant and it is quite likely that you have large amounts floating the lab, or inside your pipette barrels. You should take apart your pipettes and clean them, get new cuvettes and check that it isn't something to do with your stock of cells.

akhshik

Posted 27 December 2011 - 12:27 AM

I've been screening mutant libraries and the libraries keep ending up with the same mutant. I made new libraries and the same thing happened again. I suspect 2 sources : reused electroporation cuvette and aerosol during pipetting. I can easily use new cuvettes but I seriously doubt it is the main cause because I've tried transforming cells in the old cuvettes just to test the sterility and got no colony. Moreover, I exposed the cuvettes to UV at 300nm over the illuminator for DNA gel for 10min before using them. I guess that should damage whatever residual DNA that could have transformed. I'm thinking if aerosol generated by pipetting during cell preparation could have caused the cross-contamination. However, instinctively, I find it hard to believe the (invisible) aerosol could cause such extensive contamination. Has anyone experienced cross-contamination due to aerosol generated by pipetting?


Samplers are the main source for every contamination, did you used filters, or not?

phage434

Posted 27 December 2011 - 08:43 AM

1 M HCl for a few minutes followed by good rinsing is far more effective at removing DNA than UV exposure, which probably misses many of the places inside a cuvette.
Bleach (10%) also works quite well.