Team:Utah State/Notebook

Transformations are any procedure used to insert DNA into a bacteria (if you use a virus, the term becomes transfection). Electroporation uses a pulse of electricity to disrupt the cell membrane and create holes that would allow the DNA to enter the cell. Cells need to be made competent before doing this procedure, in order for them to efficiently take up the DNA. Transformations generally utilize millions to billions of cells and DNA molecules and, for a transformation to be successful, only one molecule of DNA needs to enter into one cell, which then grows into a colony. One issue with transformations is selecting and verifying which colonies have the desired DNA. This is usually done using a marker, a characteristic possessed by cells that have the DNA (or lost by those cells) that distinguishes it from the rest of the colonies that grow up. Commonly, this is the expression of an antibiotic resistance gene included on the transformed DNA, which allows only the cells that have taken up the DNA to survive on a plate in the presence of that antibiotic. Sometimes pigment producing or fluorescent/luminescent proteins can also be used in place of antibiotic resistance to allow visual determination of transformed colonies. Other ways of selection exist, but will not be discussed here.

1. Turn on ice machine
2. Thaw DNA solutions
3. Clean and sterilize the electroporation cuvettes by washing with double distilled water (ddH2O) twice and then fill the cuvettes with ethanol.
4. Let cuvettes sit with ethanol for 5-10 minutes, then wash 4-8 times with ddH2O
5. Place cuvettes on ice
6. Take competent cells out of the -80 °C freezer, and thaw them on ice
7. Add 3 µL of DNA to the cell solution. (This should be around 100-250 ng of total DNA, too much DNA causes arcing, too little gives few transformed colonies).
8. Incubate on ice for 5 minutes.
9. Add 60 µL WB buffer (10% glycerol). This helps reduce arcing, although too much can lower numbers of transformed colonies.
10. Set the electroporation machine to 2500 V, 200 O, and 25 µF for E. coli.
11. Transfer the cell/DNA/WB solution into the cuvettes by pipetting up and down in the 1.5 mL tube first to mix. Make sure the pipette tip is between the metal plates on the cuvette before ejecting the solution. Keep the cuvettes on ice.
12. Before electroporating, dry the cuvettes of with a KimWipe, to ensure no liquid on the surface that could create other paths for the electric pulse (and could cause arcing).
13. Pulse the cells and return cuvette to the ice. Check the time constant on the machine, a constant of 4.5+ is a very good transformation, and will yield many colonies. A constant of 2.5-4.5 is okay, and will still work. Constants below 2.5 will yield very low colony numbers, and may need to be redone. NOTE: addition of extra WB or lower amounts of DNA will reduce the time constant as well, so it is only a rough measure.
14. To remove the cells from the cuvette add 1 mL LB media or SOC media (no antibiotic in this media) to the cuvette. Pipette up and down a few times to mix.
15. Remove the solution to just above the two plates in the first removal pipetting (~1/2 of the volume) and transfer it to the original cell tube (NOT THE DNA TUBE). Then, tip the cuvette on its side so that the space between the plates is vertical, place the 1000 µL pipette tip between the plates, and slowly draw up the solution, while tipping the cuvette further. This should draw up the rest of the liquid in the cuvette.
16. Incubate the cell solutions at 37 °C for 1-2 hours (can go up to three, but try to avoid doing it for that long).
17. Plate the cells on plates containing the correct antibiotic. Each transformation requires two plates. Add 500 µL of solution to one plate, spread with the spreading stick, and then spread the spreading stick on the second plate without adding any solution to it. This creates a dilution plate in case you have thousands of colonies on the first plate. It is roughly a 1:100 to 1:200 dilution.
18. Grow the plates up overnight at 37 °C. Do not leave for longer than 24 hours, as contaminants might have a chance to grow and the plates could dry out.

If your cuvette arcs (bright flash and loud popping noise during electroporation):

1. Clean the electroporator lid.
2. Wash out and sterilize the cuvette with ethanol – the cells have been pretty much killed and will not be usable in plating, so you need to restart.
3. Add less DNA to the cells (reduce by 25%-33%).
4. Add an additional 15 µL of WB buffer to the solution.