Burkart:Competent Cell: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
| Line 18: | Line 18: | ||
==Protocol== | ==Protocol== | ||
1. Prepare fresh overnight 2 mL LB (with antibiotics if already containing plasmids) culture from plate colonies or frozen stocks. | |||
2. Inoculate fresh overnight culture to 50 mL LB media in 1:250 (200μL) or 1:500 (100μL) dilution. | |||
3. Incubate at 37°C with agitation until the Optical Density (OD<sub>600</sub>) reaches 0.4-0.5 (about 3hr). | |||
4. Transfer the culture into 50 mL centrifuge tube and chill the culture on ice for 10-15 min. | |||
==Note== | ==Note== | ||
Revision as of 09:37, 27 April 2009
RbCl Competent Cell Preparation
Introduction
Materials and Equipments
RF1 buffer
RbCl 100mM (12g/L)
MnCl2·4H2O 50mM (9.9g/L)
Potassium Acetate 30mM (2.9g/L)
CaCl2·2H2O 10mM (1.5g/L)
Glycerol 15%(w/v)
Adjust pH to 5.8 with acetic acid. Sterilize.
RF2 buffer
RbCl 10mM (1.2g/L)
MOPS 10mM (g/L)
CaCl2·2H2O 75mM (11g/L)
Glycerol 15%(w/v)
Adjust pH to 6.8 with NaOH. Sterilize.
Protocol
1. Prepare fresh overnight 2 mL LB (with antibiotics if already containing plasmids) culture from plate colonies or frozen stocks. 2. Inoculate fresh overnight culture to 50 mL LB media in 1:250 (200μL) or 1:500 (100μL) dilution. 3. Incubate at 37°C with agitation until the Optical Density (OD600) reaches 0.4-0.5 (about 3hr). 4. Transfer the culture into 50 mL centrifuge tube and chill the culture on ice for 10-15 min.
