I am trying to Electroporation of Synechosystis cells using 2.5 Kv ( 12.5 KV/ Cm) voltage in 2mM, 1 mM HEPES (pH 7.5)or 5 mM NaCl. I washed the cells three times before set for Electroporation using the above buffer. Then 40 microL cels with 2 microL DNA has been taken and placed in the cuvette ( 2 cm) , operate with the condition Kv 2.5 in manual option. But after pressing pulse, it is Arcing and dispersing the sample inside of the cuvette. The Milisecond ( ms) constant is showing 0.00 which should be around 5 according to the manual of Electroporation. However, in lower KV such as 1.5 it is showing around 5. Now the question is that is that transformation acceptable in lower voltage or how can I increase the Ms constant using higher voltage?
Salt carry over can be a problem from BG11 medium used for growth of your cells. Washing them additional times may help. Ensure the DNA is "clean" also as Yordan suggests
Be sure your DNA is well purified and contains no residual salts. Good luck!
Salt carry over can be a problem from BG11 medium used for growth of your cells. Washing them additional times may help. Ensure the DNA is "clean" also as Yordan suggests
Nothing to add in particular to the already given suggenstions. Get rid of the salts, that might be your major problem. The settings seem ok, though.
As long as your time constant is up it should be ok - I had the same experience with cyanos at lower voltages, 2.5 KV always arced and 1.4-1.5 KV gave me time constants up to 5 ms.
Anyway, there is this protocol: http://2008.igem.org/Team:Hawaii/PCC6803_Electroporation_of_PCC6803 where "60 uL of cell suspension mixed with 0.1-16 ug DNA (dissolved in H2O) and electroporated with Biorad gene pulser (25uF capacitor used, time constant varied by changing resistors (100, 200, 400, & 600 Ohms for time constant 2.5, 4.8, 9, 13 ms, respectively, electric field varied from 0-12kV cm-1)" - I don't think changing the resistor will change much, they claim "40uL of cells at 10^9 cells/mL were mixed with 5 uL of plasmid DNA solution (1ug/uL). Electroporation was performed at field strength of 12kV/cm at a constant of 5ms, with no cell death detected." as you were expecting, I would try varying the DNA concentration as they did. I'm curious what transformation efficiencies you will achieve at the different settings...
@Christian Tagwerker ,
Thanks for your information. I would like to know that " did you get positive transformant at lower voltage like 1.4 to 1.5 with higher ms. What types of Cyanobacterial cell you used? How did you solve that problem of arching at higher voltage?
Thanks
Is your cuvette the right size? With 0.1 cm gap cuvette 1.25 kV pulse should be applied (25 uF, 200 Ohm). With 0.2 cm gap size cuvette you would apply 2.5 kV pulse (again 25 uF, 200 Ohm).
If you are getting arcing then changing voltage will not fix anything. Arcing almost always means you have salt in the cuvette, either from the DNA or from the cells.
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