Trying to transform without succes - (Feb/22/2007 )

Hello,

I am trying to transform by heat shock (42 C per 1 min) DH5alfa competent cells with a bacterial plasmid ligate with an Arabidopsis insert. I'm making the transformation with 50 ul of competent cells and 2 ul of the ligation. At first I used LB nutrient media, now I'm using SOC for increase the transformation but I didn't obtained any colony. What changes you reccomend to made my protocol with succes? What factors increase the efficency os a transformation? How I could verify if the problem is the competent cells?




Thanks,
lirio de la rivera

Hi
I am not an expert but I can try to help according to my experience.
Many factor can be the problem.
Did you checked that the arabidopsis fragment you want is actually inserted in the vector?
Other thing that seems that really matter is the incubations times...the longer the better.
when you prepare the vector you need to incubate at 25 C I think. Most protocols say less than 30 min...I let it an hour.
You need to be careful that you are not using too much or too less from the pcr product when inserting it in the vector. around 0.7 microg/microl is ok.
I usually used all the vector mix (6 microl)for 50 microl of Top10 (another e. coli strain). I let the E. coli mix on ice for 30 min and then heat shock 42 C for 30 sec.....ice 1 min. and then 250 microl of SOC. I incubate 1 hour and spread on kanamicin plates. It works ok.

I hope it can help

Good luck

Solmaniar:


Thanks a lot for your recommendations. Yes, I'm sure the ligation its fine. I will triynig changing the times and the quantities as you said.


Peace,
lirio de la rivera

good luck...