transformation problems - (Jun/11/2005 )

hi
I am doing some site directed mutagenesis experiments and am in the process of transforming my mutated products.My recombinant plasmid has ampicillin resistance while my competent cells have both tetracycline resistance and chloramphenicol resistance.When I plated my recombinants,I got the following results:
1)negative control and sample plates had colonies.
2)sample plates had less no of colonies as compared to the neg control.
3)the colonies grew towards the outer rim of the plate.
Can I Assume that: 1)antibiotic is not working?
2) competent cells are not working;-how can I f ind this out?

Thanks
jyothi

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Dear Jyosaj,
Can I ask you something?
Does your agar plate contains all three antiboitics during sellection?
What do you means by "colonies grew towards the outer rim of the plate"? Is there any colonies growth in the middle of the plate?
What is your final concentration of each antibiotic added?

Regards
Hadrian.

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what kinds of antibiotic does your plate has?

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HI Hadrian,
My selection plates contain ampicillin only,for selection of recombinants.I didnt put tet/chlorampenicol.
The colonies were absent in the middle of the plate.they grow only in the area just after the rimo f the plate. So I am not sure whether it is due to antibiotic defect combined with some contamination.
final concentration of ampicillin 100 microgram/microlitre.

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I am thinking that I will just plate some of the original stock of comp cells on fresh LB agar with no antibiotic and all the three antibiotics separately and see what happens? I can also autoclave my stuff in anotehr machine to see whether there is any prob with the autoclave machine. Recently,I have also observed my media get contaminated very fast.Earlier,it was not like that.

What do you suggest?

Jyosaj

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