Hello! I am trying to use BTX ECM830 electroporator to introduce plasmid DNA into mammalian cells. I have tried using DMEM, HEPES-Based buffer and PBS, without success. i have used between 50 and 150 volts and 30ms. Every time I get ARC and the electroporation aborts. i get ARC even using 10 V. I am using 2mm couvette.
How can I set the parameters to not get ARC? Can I electroporate with just Glucose or trehalose?
Many thanks!
Here are the steps on how to introduce plasmid in mammalian cell electroporation using BTX EXM 830 without getting ARC:
1. Prepare the cells. The cells should be in logarithmic growth phase and should be at a density of 1-5 x 10^6 cells/mL.
2. Prepare the plasmid DNA. The plasmid DNA should be dissolved in a buffer that is compatible with the cells. The concentration of the plasmid DNA should be 1-10 ug/mL.
3. Set up the electroporation cuvette. The cuvette should be filled with the cell suspension and the plasmid DNA. The ratio of cells to plasmid DNA should be 10:1.
4. Choose the appropriate electroporation parameters. The electroporation parameters will vary depending on the type of cells being used. The following are some general guidelines:
* Voltage: 100-200 V
* Pulse width: 1-10 ms
* Recovery time: 1-5 min
5. Perform the electroporation. The electroporation should be performed in a cold room. The cuvette should be placed in the electroporation chamber and the voltage should be applied. The pulse width and recovery time should be as specified in step 4.
6. Incubate the cells. The cells should be incubated at 37°C for 1-2 hours.
7. Add selection antibiotic. If the plasmid DNA contains a selection marker, such as an antibiotic resistance gene, then the cells should be incubated with the appropriate antibiotic for 24-48 hours.
8. Assay for transfection efficiency. The transfection efficiency can be determined by a variety of methods, such as the following:
* Cell counting
* Western blotting
* Flow cytometry
By following these steps, you can introduce plasmid in mammalian cell electroporation using BTX EXM 830 without getting ARC.
Tips to help you avoid ARC:
Use a low voltage and pulse width.
Use a recovery time of 1-5 minutes.
Incubate the cells at 37°C for 1-2 hours.
Add selection antibiotic.
Assay for transfection efficiency.
By following these tips, you can increase the success rate of your electroporation experiments and avoid ARC.
Yes, you can electroporate with just glucose or trehalose. However, it is important to note that the efficiency of electroporation will be lower than if you use a buffer that contains other ingredients, such as serum or HEPES. This is because glucose and trehalose are not as effective at protecting cells from the damage caused by electroporation.
If you choose to electroporate with just glucose or trehalose, you should use a lower voltage and pulse width than you would if you were using a buffer with other ingredients. You should also incubate the cells for a longer period of time after electroporation.
Tips for electroporating with just glucose or trehalose:
- Use a low voltage and pulse width.
- Incubate the cells for a longer period of time after electroporation.
- Add selection antibiotic.
- Assay for transfection efficiency.
By following these tips, you can increase the success rate of your electroporation experiments.
Glucose is a simple sugar that is found in many foods, including fruits, vegetables, and grains. It is also a major component of blood sugar.
Trehalose is a disaccharide that is made up of two glucose molecules. It is found in many fungi, insects, and plants. Trehalose is a good protectant against freezing and desiccation.
Hello John,
Many thanks for your promt answer! I will try those parameters. I have tried using DMEM, SFB, HEPES, salts, PBS, and all of them, even at low concentrations gave ARC, I also tried 10V-100V and also ARC. Which buffer will you recommend?
Much appreciated!
Salts are often used in buffers to adjust the ionic strength. PBS is a good choice for buffers that need to be inexpensive and easy to prepare. HEPES is good choice buffers that need to be stable over a wide pH range. Serum-free basal medium is best buffer to avoid contamination. DMEM (Dulbecco's Modified Eagle Medium) is not a good buffer.
SFB is a 3D filament, not the best choice. But it does prevent contamination better. MES buffer, or 2-(N-morpholino)ethanesulfonic acid, is good wide range of proteins and nucleic acids. TAE buffer is a mixture of Tris base, acetic acid, and EDTA. TAE buffer is good for Agarose gel electrophoresis
RNA electrophoresis
DNA sequencing
Protein electrophoresis
PIPES buffer is often used in cell culture, protein purification, and enzyme assays.
The best buffer for electroporation will depend on the type of cells you are using and the type of DNA you are transfecting.
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