Dear colleagues,
I have a question regarding bacteria transformation.
I am working with Agro-bacteria GV3101, and want to transfer the plasmid into it.
When I get a pure plasmid from cloning, can I transform them into Agro-bacteria directly? Should I transform to competent E Coli first?
And second question, my plasmid construct is quite big, about 15Kb. I am aware of large plasmid, it is better to use electroporation. However, my lab does not have an electroporator, can large plasmid transformation still work by heat shock?
and final question, before transformation, how should I store pure plasmid?
Thanks for the help.
Kind regards,
Rex
You should definitely transform it into E. coli first. For one because then you have a back up you can use for plasmid preparation and because you should sequence after clone, to make sure that there aren't any point mutations or whatsoever.
Transformation by heat-shock will be much less efficient, but it should work eventually. You might need to use a lot of DNA for the transformation.
We store our plasmid in pH correcter water (pH 8.5) or Tris-HCl buffer at -20°C. As long as you have a buffered solution it should even be alright to store them at 4°C for a short time period.
Simon Flueckiger Hi Simon, appreciate for your prompt reply. How much DNA do you think I need for bacteria transformation. I plan to construct my plasmid by Gibson assembly, I am not sure if this will generate enough DNA.
In addition, for plasmid storage, what is the ratio of corrector and plasmid DNA?
Thanks.
Rex
Hi, My first impression is that normal CaCl2 mediated bacterial transformation should work with the sized plasmid of yours. However there are few factors which you should be considerate of to enhance/influence the uptake of plasmid by the cells.
1.Use freshly made competent cells because if they are old and have undergone several freeze thaw cycles, the yield might be affected.
2. Keep the cell on ice for 4-5 min after heat shock (HS should be 45 sec at 42 C).
3. Use the SOC medium instead of normal LB.
4. After incubating at 37 C, spin down the cells before spreading on plates (Suppose u have 250 ul in total, so spin down and throw the 200 ul supernatant and proceed with 50 ul cell suspension to spread on the plate screened for appropriate antibiotics).
Additionally make sure the plasmid you are using is free from any solvent contaminant from the upstream mini/maxi prep plasmid isolation step.
Hope it works, and still if doesn't then just opt for STBL2 cells which are high-efficiency chemically competent cells specifically designed for cloning unstable and large inserts.
Harvijay Singh thanks for your reply. The competent E Coli cell is included in the Gibson Assembly kit, so I assume it is fresh.
But the Agrobacteria GV3101 I have was leftover from previous students. It did not thaw/freeze for many time, but it is about 1 year old. Do you think if this might be the issue?
Eventually, I want to transform the plasmids into Agrobacteria. In my understanding, I should transform competent E Coli first. Then isolate plasmid from E Coli again and then transform into Agrobacteria. Is this correct? The Agrobacteria GV3101 is not chemically competent, does this matter?
Thanks for the help.
Rex
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