TITLE: Electroporation Protocol (C2986)
AUTHORS: New  England   Biolabs

[DESCRIPTION]
NEB Turbo Electrocompetent E. coli cells are suitable for high efficiency electroporation and rapid colony growth. These cells are ideal for DNA library constructions and all cloning purposes.

[GUIDELINES]
Features
Electroporation Tips
Electroporation cuvettes and microcentrifuge tubes should be pre-chilled on ice. 
Electrocompetent cells should be thawed on ice and suspended well by carefully flicking the tubes. 
Once DNA is added to the cells, electroporation can be carried out immediately. It is not necessary to incubate DNA with cells. The maximum recommended volume of a DNA solution to be added is 2.5 µl. Addition of a large volume of DNA decreases transformation efficiency. 
Contaminants such as salts and proteins can lower electroporation efficiency. Ideally, DNA for transformation should be purified and suspended in water or TE. Transformation efficiency is more than10-fold lower for ligation mixtures than the control pUC19 plasmid due to the presence of ligase and salts. If used directly, ligation reactions should be heat-inactivated at 65°C for 20 min and then diluted 10-fold. For optimal results, spin columns (NEB #T1030) are recommended for cleanup of ligation reactions. 
Electroporation conditions vary with different cuvettes and electroporators. If you are using electroporators or cuvettes not specified in the protocol, you may need to optimize the electroporation conditions. Cuvettes with 1mm gap are recommended (e.g. BTX Model 610/613 and Bio-Rad #165-2089). Higher voltage is required for cuvettes with 2 mm gap. 
Arcing may occur due to high concentration of salts or air bubbles. 
It is essential to add recovery medium to the cells immediately after electroporation. One minute delay can cause a 3-fold reduction in efficiency. 
Cold and dry selection plates lead to lower transformation efficiency. Pre-warm plates at 37°C for 1 hour. Using 37°C pre-warmed recovery medium increases the efficiency by about 20%. 
Refreeze unused cells in a dry ice/ethanol bath for 5 min and then store at -80°C. Do not use liquid nitrogen. Additional freeze-thaw cycles result in lower transformation efficiency.

Application Features
DNA Effects of Transformation Efficiency and Colony Output:
  


[STEPS]
SECTION: Preparation
1. Prepare 17 mm x 100 mm round-bottom culture tubes (e.g. VWR #60818-667) at Room temperature.
SECTION: Preparation
2. Place SOC recovery medium in a 37 °C water bath.
SECTION: Preparation
3. Pre-warm selective plates at 37 °C for 60 min.
SECTION: Electroporation
4. Place electroporation cuvettes (1 mm) and microcentrifuge tubes on ice.
SECTION: Electroporation
5. As a positive control for transformation, dilute the control pUC19 by 1:5 to a final concentration of 10 pg/μl using sterile water.
 

SECTION: Electroporation
6. Thaw NEB Turbo Electrocompetent cells on ice (about 10 min ) and mix cells by flicking gently.
SECTION: Electroporation
7. Transfer 25 µL (or the amount specified for the cuvettes) to a chilled microcentrifuge tube.
SECTION: Electroporation
8. Add 1 µL.
SECTION: Electroporation
9. Carefully transfer the cell/DNA mix into a chilled cuvette without introducing bubbles and make sure that the cells deposit across the bottom of the cuvette.
SECTION: Electroporation
10. Electroporate using the following conditions for BTX ECM 630 and Bio-Rad GenePulser electroporators: 
     2.1 kV, 100 Ω, and 25 μF

The typical time constant is ~2.6 milliseconds.
SECTION: Electroporation
11. Immediately add 975 µL to the cuvette.
SECTION: Electroporation
12. Gently mix up and down twice.
SECTION: Electroporation
13. Transfer to the 17 mm x 100 mm round-bottom culture tube.
SECTION: Electroporation
14. Shake vigorously (250 rpm) or rotate at 37 °C for 60 min.
SECTION: Electroporation
15. Dilute the cells as appropriate, then spread 100 µL-200 µL onto a pre-warmed selective plate.
SECTION: Electroporation
16. Incubate plates 480 min to   at 37 °C.