I have a large plasmid (20kb) which I am trying to transform into chemically competent commercial EHA101 cells. I had success with transforming a 16kb plasmid into the same strain, but have been unsuccessful with my larger plasmid.
This was my method:
1) Thawed agro cells from -80 in hand
2) added 2.5ug (5uL) of each pGE013_Upf1sgRNA_1
3) 30min on ice
4) 5min in liquid nitrogen
5) 5min in 37 water bath
6) 5min in ice
7) Add 900uL of YEP
8) Incubate at 28C with shaking for 7 hours
9) Centrifuge at 7000rpm and remove 900uL of supernatant
10) Resuspend cells in remaining supernatant
11) Plate on YEP+spec and YEP +spec +kan
Wrap with parafilm and incubate at 28C. Saw growth for 17kb plasmid (binary CRISPR/Cas9 vector) after 4 days on YEP+kan+spec. These colonies grew on YEP+spec+kan+rif. I also purified plasmid from these via alkaline lysis and saw it present on agarose gel but no growth on the 20kb plasmid plate (pMpGWB337 vector with insert).
I had previously tried a shorter incubation of the cells+plasmid and a shorter outgrowth period.
I don't have the materials for electroporation, so I am very hopeful that I can somehow make the freeze-thaw method work.
Many thanks!
Can always try calcium phosphate nanoparticles.
With chemically competent cells you usually need to heat shock them at 42 degrees for a few mins before chilling on ice and then adding your rich media.
Thanks for the response, Robert. Are you suggesting a 42 C incubation instead of the 37 C?
Yes, the freeze-thaw method can be used for the transformation of Agrobacterium with large plasmids. This method involves subjecting Agrobacterium cells to multiple freeze-thaw cycles, which creates small pores in the cell membrane, allowing the plasmid DNA to enter the cells.
@Hassan I would like to try this. Can you share more specific instructions? And how many cycles you recommend trying
Lauren Woodward Hartzler
Competent Agrobacterium cells:
Transformation:
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