5 replies to this topic

[deespike]

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?

[pito]

deespike, on 13 September 2010 - 07:23 AM, 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.

[microgirl]

The plasmid probably has both an amp and a kan resistance cassette.

[microgirl]

Sounds like your competent cells may be contaminated with something that's kan resistant. Try a new competent cell prep?

[Ameya P]

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.

[deespike]

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.