Preparing cells for electroporation

1. Culture CH₁₂F₃-2 cells such that they are in the logarithmic growth phase on the day of electroporation. Typically, seeding 1 million cells in 10mL CH₁₂F₃-2 media the day before works well. You will need 1 million cells per electroporation.

Electroporation

2. Resuspend 1 million CH₁₂F₃-2 cells in 600µL ZAP buffer, and combine with 5µg CRISPR construct in 4 mm electroporation cuvette.

3. Electroporate in BioRad GenePulser Xcell with following conditions:

4. Immediately place electroporated cells on ice and incubate for 5 min, then plate in 10mL CH₁₂F₃-2 media and incubate @ 37 Celsius/5% CO₂.

CRITICALImmediately place electroporated cells on ice and incubate for 5 min, then plate in 10mL CH₁₂F₃-2 media and incubate @ 37 Celsius/5% CO₂.

5. Resuspend 2 x 10⁶ cells in 100µL of electroporation solution and add to a 2 mm electroporation cuvette

6. Add 5µg of your Cas9 and gRNA plasmid.

7. Electroporate cells with 250 volts for 5 msec

8. Immediately transfer the cells from cuvette into 1mL of culture media after electroporation

CRITICALMinimize the time between electroporation and transfferring the cells to increase cell viability.

72 hrs after electroporation

9. Serially dilute electroporated cells to 10-20 cells/mL and multi-channel pipette 100µL into 96-well flat bottom plates. Typically, 10 x 96-well plates @ 20 cells/mL yields around 100 clones, perfect for a 96-well PCR block. Sorting single cells should also work, but is more expensive!

An accurate and precise count with a hemocytometer is required; for example, a count of a dozen cells in one field is not accurate. Do not serially dilute at more than 1:100 per dilution.

While in theory 10 x 96 wells @ 0.2 cells/well (20 cells/mL) should result in 192 clones, you will get less likely due to adsorption of cells to plastic; however, you will reproducibly get approximately 100 clones. You can screen less clones, but 96 clones is definitely manageable starting off, and screening two constructs in parallel (2 x 96 clones) is feasible with practice.

Note that a 72 hour rest post electroporation may not be optimal for all genes, especially for genes where deficiency causes a growth defect.

10. Incubate clonally plated cells in the long-term 37 Celsius incubator, to reduce water loss, for 7 to 10 days. Clones should be observable by microscope @ 4-5 days, and by eye @ 5-7 days. There is some timing flexibility with step 24 and onwards.