酵母电转化参数

Protocol 0342

ElectroSquarePorator

PROTOCOL

T820 ELECTROPORATION

Cells: E. coli XL1-Blue or DH5a

Plasmid: pBluescriptKS (Stratagene)

Cell Preparation:

Growth medium: Grow cells in SOC to OD600=0.6

Washing procedure: Wash the cells with 200ml SOC in a 2L flask. Spin the cells @ 3000 rpm for 5-10

min. each time. Resuspend in 10 ml of ice cold ddH2O (must use ice cold

ddH2O, or 1 mM HEPES @ pH 7.0. The efficiencies are comparable.). Aliquot

into 50-200 µl samples.

Note: It’s very important to prepare competent cells carefully to not lose their competency. Both aeration and temperature control are critical. The higher quality water you use for preparing the SOC and for rinsing, the better the efficiency will be. Try to use ddH2O for everything if possible. The author suggests that the cells be thawed out immediately prior to electroporation. He also recommends that the DNA be diluted in H2O just before use. He uses 0.1ng of DNA in 0.1 µl of H2O to keep the DNA concentration @ 1 µg/ml. This is a critical point. (If SOC is not available, it can be substituted with LB plus Mg++.) Electroporation Settings:

Mode: LV

Choose

V

500

Set

Voltage:

Set Pulse Length: 8 msec

Set Number of Pulses: 1 pulse

Chamber: BTX Disposable Cuvette 1 mm gap, P/N 610

Desired Field Strength: 5 kV/cm

Electroporation Procedure:

Sample Volume: 50 µl

ng

1

DNA

Amount:

Temperature: Prechill the cuvette on ice for 1 min.

Pulse: Press the Start button to activate the Automatic Charge & Pulse sequence Plate: After electroporation, immediately (< 5 sec) add 1 ml of warm SOC (37o C) to the

cuvette. Transfer the sample into tubes and incubate for 45 min. @ 37o C on a

shaker.

Results: 2.5 x 109 cfu/µg DNA

Reference: Personal communication, Dr. Masaya Yamamoto and Ha Do, University of Texas

SW Medical Center, Dallas, TX

©2000, BTX Division of Genetronics, PR0342