gene transfer via agrobacterium - how to prepare bacteria??? (Feb/15/2007 )

Hello everyone!

Here in the lab, we want to make genetic transformations using Agrobacterium tumefaciens technique
of plant tissues with a new plasmid we bought.
Soon I will have my brand new plasmid and my brand new bacteria, but...

...I really don't know which are the possible strategies to insert the plasmid in the bacterium, and I don't know how to do it and I'm going to panic soon...
So I need all your reccomendations to understand how to design my next steps.
Any suggestion will be of great help
Thanks in advance!!!
ILA

Hi...

The way I transform agrobacterium is

-Mix 100 microl of agrobacteria whit 3 microl of the plasmid.
-place the mix in liquid nitrogen for 5 min.
-and the incubate at 37 C for 25 min
-add 1 ml of YEP medium to the mix and incubate for 2 hours at 30 C
-centrifuge 2 min at 3000 g
-take most of the medium out of the tube, just leave around 50 microl and by pipeting resuspend the pellet
-spread the 50 microl in an LB plate with rifampicin 50mg/l and gentamycin 25mg/l..and wait for around 2 days.
( you can use the colonies for transform Arabidopsis, and do not forget to make some frozen stock (-80 C))

Before doing it check with other protocols... I used this one 2 times and it worked ok.

Good luck

Thanks very much!!!

Take a look to this site: http://www.bioinformatics.vg/Methods/agrotransformationf.htm
It's a very complete protocol and they also explain how to prepare competent cells that you can use at a later date.

Good luck!!

hi

hope i am not too late

There are two types of transformation
freeze thaw method and electrophoresis

if going for electrophoresis, the transformation is very efficient, but the competent cells have to be prepared without salt presence preferably with HEPES or Glycerol

IN freeze thaw , ( personally i used this one since i dont have to bother any one else , the electrophoresis machine is in another lab) you have to use a -70 et OH bath to freeze the cells.. easy to make , just put some etoh in a closed container and keep in deep freezer for 5-16 hours, here the competent cells can be made by CaCL2 method.


" Efficient transformation of Agrobacterium spp. by electroporation"
Diethard Mattanovich et al, Nucleic Acid Research , Volume 17,number 1,1989


Biotechniques 16(4) 1994, 664,669

An,Gynheung (1987) Methods in Enzymol. 153;292-305




hope this was helpful

best of luck

dear all,

I tried to transform my plant with wildtype Agrobacterium rhizogenes A4. The type of explant I used is leaf. It supposed to produce hairy roots. However, the leaves only induced normal roots. At first, I thought it was because of the bacteria problem. But after I checked the plasmid with PCR, the rolB gene was present! Therefore I think the bacteria is not a problem. So, is it possible to be the plant itself is resistant towards the infection of Agrobacterium? If so, what steps can I take to overcome the problem? Does the culture medium and condition affect the transformation method? Thanks!