contamination in kanamycin plate - (Sep/13/2010 )
HI all
i have been doing transformation with a kanamycin resistant plasmid into dh5alpha cells , but each time i get white colonies and not the translucent type dh5alpha colonies, to check them i grew them in ampicillin and there was no growth so i confirmed that its having the kanamycin resistant plasmid. After isolating the plasmid i found the conentration to be very less .
Can anyone plz suggest how to get rid of these colonies growing over dh5alpha?
deespike on Mon Sep 13 15:23:48 2010 said:
HI all
i have been doing transformation with a kanamycin resistant plasmid into dh5alpha cells , but each time i get white colonies and not the translucent type dh5alpha colonies, to check them i grew them in ampicillin and there was no growth so i confirmed that its having the kanamycin resistant plasmid. After isolating the plasmid i found the conentration to be very less .
Can anyone plz suggest how to get rid of these colonies growing over dh5alpha?
Just a quick note: you tested the cells by growing them in ampicillin to confirm they are having the kanamycin resistant plasmid? I do not understand this.
The plasmid probably has both an amp and a kan resistance cassette.
Sounds like your competent cells may be contaminated with something that's kan resistant. Try a new competent cell prep?
Well, you cant say that cells are Km resistant when they do not grow over Amp.
I think your Km is too old and not very effective thereby allowing other cells to grow.
If not, then your competent cells are contaminated.
So, get Km from a different lab and plate your cells. You should then just get cells which contain the plasmid.
Thanks for your replies.
I will test kanamycin again. I have tested the competent cells they are not contaminated but still as the same thing happens so i will change the kanaycin.