Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Cloning

To what extent competent cells be thawed before transformation? - (Mar/03/2009 )

Hi,

Recently I have not been getting colonbies on my plate, tried out new ligase, new ligation buffer, different vector to insert ratio etc but just 3-6 empty colonies per plate. Is there a way to thaw frozen competent cells? I usually haw them on ice and when they are completely thaw, I add the ligaion mixture. I heard some people add teh ligation mixture when hey are semi-thawed?

-SF_HK-

I thaw cells on ice for 10-15 min. depending if I have time, or simply add the ligation mixture when it is semi thawed, its the same thing. The idea is to mix cells and ligation mixture.

-scolix-

I have a stupid question for you:
Is the selective gene the same as the antibiotic?

I once tried growing Kanamycin resistant on an ampicillin plate (with out much success).
I learned a good lesson from it never take for granted what people tell you, always check it your self!

-molgen-

molgen on Mar 8 2009, 08:51 AM said:

Is the selective gene the same as the antibiotic?


No. The "selective gene" is a gene that confers resistance or sensitivity to some substance, not always an antibiotic (the "selective substance", if you like). There are genes that confer resistance to substances that aren't antibiotics, like HgCl2, and there are genes that confer sensitivity to other substances, like sucrose, that allows one to counter select.

-HomeBrew-

molgen on Mar 8 2009, 04:51 AM said:

I have a stupid question for you:
Is the selective gene the same as the antibiotic?

I once tried growing Kanamycin resistant on an ampicillin plate (with out much success).
I learned a good lesson from it never take for granted what people tell you, always check it your self!


i am continuing the work of my previous colleague, but her data already pissed me off becoz most of her works were not very reproducible. Good point by molgen.

-Nrelo-