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now: Wed Sep 19 18:07:29 2018 ... mod: Mon Oct 12 04:41:26 2015
What do I think, Dear Author? I think you are awesome. Once upon a time I did think idly about what it would take to do moelcular biology at home. I actually think it might be a lot cheaper than doing it in a real research institute buying a fridge from the local discount store, for example, rather than the up-priced version from the supplier catalogue.I suspect that one major challenge would be convincing suppliers that you were legit, and that they should deliver enzymes to a residential address. But that's probably a minor detail, as long as the credit card holds up. That and the inevitable complaints about the neighbours if they find out.
Electroporating Tissue Culture Cells
Electroporation conditions must have electrical parameters optimized for each cell line. In general we use complete culture medium for electroporation and 10 μg of plasmid.
- Two days before electroporation: split cells to a final density of from 10% to 20% confluency on a 10 cm dish
- for adherent cells it generally takes one day to recover from the split procedure, and one additional day for maximal log-phase growth. cells electroporate in rough proportion to how "happy" they are, where happiness is usually maximized by log-phase growth
- the cells should be abundant but not confluent the day of electroporation
- Two hours before electroporation: equilibrate both complete tissue culture medium and trypsin/EDTA solutions to room temperature
- leaving bottles out on the bench for a few hours works well
- it is critically important that neither of these solutions be cold when used on cells to be electroporated
- Electroporation time: aspirate off old medium and add 2 ml room temperature trypsin/EDTA to each 10 cm dish. treat with trypsin/EDTA until cells are about 1/4 detached from the plate with gentle agitation.
- Do not over trypsinize the cells
- while cells are trypsinizing, add 10 μg plasmid to be electroporated to the bottom of a 0.4 cm gap electroporation cuvette
- the plasmid should be a solution in either water or 1/10x TE. the plasmid should be at a concentration of approximately 1 μg/μl so as to not dilute the electroporation mixture excessively with the plasmid solution
- ensure that all cells have detached by firm but gentle pipetting of the trypsin/EDTA solution over the cells using a P1000 pipette tip
- stop the action of the trypsin/EDTA by adding 4 ml room temperature complete tissue culture medium.
- transfer cells to a 15 ml conical centrifuge tube and spin down at 200 g for 5 minutes to pellet.
- the pelleted cells are reasonably stable in the pelleted state, so you can prepare multiple tubes to electroporate at the same time
Do the following series of steps for each individual cell pellet to be electroporated one at a time from start to finish. IMPORTANT → If multiple cell pellets are to be electroporated, completely finish all of these step for one cell pellet before starting the next cell pellet.
- carefully aspirate the medium from one cell pellet. leave a small meniscus of medium covering the pellet
- do NOT aspirate to dryness. leave approximately 10 μl medium overlaying the cell pellet
- add 750 μl room temperature medium to the cell pellet and titurate using a P1000 pipette tip until cells are homogeneously resuspended
- transfer 750 μl of the cell suspension to a 0.4 cm gap electroporation cuvette containing the plasmid to be electroporated and gently but thoroughly mix by pipetting up and down
- do NOT make any bubbles in the cell suspension in the electroporation cuvette. if bubbles are inadvertently made, they must be removed prior to electroporation
- transfer only 750 μl of cell suspension. there should be some cell suspension left over in the bottom of the centrifuge tube after removal of the 750 μl due to the meniscus of media that was left overlaying the cells. it is important for reproducibility that the volume of the cell suspension electroporated in each case be exactly the same
- place the cell / plasmid suspension in the electroporation cuvette into the electroporator and initiate the optimized electroporation protocol. CRITICAL → IMMEDIATELY after the electric pulses have finished, add two aliquots of room temperature complete medium to the cuvette, rapidly and vigorously pipetting up and down after each 750 μl aliquot addition for thorough and immediate mixing. IMMEDIATELY following this mixing, dump the cell resuspension onto a fresh 10 cm tissue culture dish containing pre-warmed medium. gently swirl the plate a few times to distribute the cell suspension and IMMEDIATELY place the plate into a humidified incubator. do not disturb again until at least overnight.
- "dump" means invert the cuvette over the tissue culture plate and gently shake out the electroporated cell suspension
- without skimping on any of the steps, minimize the time between delivery of the electric pulses and when the finished plate is replaced in the tissue culture incubator
- proceeding expeditiously and consistently in this step is absolutely critical to the success and reproducibility of the electroporation
- repeat preceding steps as necessary for each cell pellet to be electroporated
- in general, do NOT add selective drugs to the medium until 24 hours after electroporation