Electroporation of E. coli (SURE) cells with plasmid DNA
1. Turn on electroporator (BTX ECM 630)
- Plug in electro cell manipulator chamber
- Turn on electro cell manipulator
- Check settings (V=2.5Kv or 2500Hv, R=200ohms, C=25μf
2. Remove bacterial cells from -80˚ C; thaw at room temp and place on ice
3. Remove cuvettes from 4C
4. Dilute purified DNA, if necessary
- Plasmid DNA =100pg/ul or 0.1 ng/ul
- Ligation reaction = 100ng of total DNA (ligation efficiency < 1%)
5. Place 1ul of diluted DNA in a microtube
6. Gently aspirate 55ul of cells, and expel part of this into the microtube containing DNA then re-aspirate
- Avoid bubbles as this will help prevent arching
7. Expel the cells, and DNA, down one side of the chilled cuvette
- Tap cuvette on hard surface to distribute suspension evenly
8. Wipe moisture from the metal portion of the cuvette
- Avoid touching the metal portion of the cuvette with hands as this will warm and deposit oils
9. Place the cuvette in the chamber
10. Shock the cells once
- Electrical arcing can indicate to much salt (repeat with less DNA or fewer bubbles)
- Time constants should be 5.1-5.5
11. Place cells on ice
12. Using a sterile pasteur pipet aspirate ~1ml of 2X YT medium (r.t) and expel part of this into the cuvette; then re-aspirate
- Do not pipette up and down
13. Expel suspension down the side of a microtube
14. Place microtube in a 37C water bath for 40 minutes
15. Remove 2X YT plates from the refrigerator, and place in 37C incubator
16. Rinse cell spreader with ethanol and allow to air dry
17. Plate 100ul of bacteria (plasmid)
- Alternatively (for ligation reaction) spin tubes at 8000 rpm for 1 min.
- Pour off all but 100ul of liquid
- Gently resuspend pellet using a flicking action and vortexer
- Plate 10ul and 100ul respectively
- For 10ul plates, add 80ul of SOC prior to plating bacteria (SOC will aid growth of these colonies allowing them to grow faster which is helpful because of the small amount plated)
18. After spreading plates make sure they are dry before placing at 37C
