Burkart:Competent Cell: Difference between revisions
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==Note== | ==Note== | ||
<p>1. The growiing phase (O.D.<sub>600</sub>) of 50 mL culture is critically important to the quality of competent cells.</p> | |||
<p>2. From the step 4 on, keep every process on ice and do it quickly. High temperature and long processing time will decrease the cell quality.</p> | |||
<p>3. For saving time, ask somebody to help you opening, recapping and freezing the microcentrifuge tube while aliquoting the cell suspensions.</p> | |||
<p>4. Test your competent cell efficiency (c.f.u.) with 1 pg of plamid DNA by conventional Heat-Shock transformation. The effeciency of a good-quality competent cell should be higher than 10<sup>7</sup> c.f.u.</p> | |||
Revision as of 10:41, 27 April 2009
RbCl Competent Cell Preparation
Introduction
Materials and Equipments
50 mL LB media in a 250 mL culture flask
pre-cooled (4°C) RF1 and RF2 buffers
pre-cooled (-80°C) steriled microcentrifuge tubes
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. Store in light-proof environment.
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. Store in light-proof environment.
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.
5. Pellet the cells by centrifugation at 1000×g for 12 min at 4°C. Drain the pelleted cells thoroughly by inverting the tubes, and use pipette to draw off recalcitrant drops of media.
6. Resuspend the cell pellet with 15 mL of pre-cooled RF1 buffer. Do it on ice and quickly.
7. Incubate the cell suspension on ice for 15 min. And pellet cells as in step 5.
8. Resuspend the cell pellet with 4 mL of pre-cooled RF2 buffer. Do it on ice and quickly.
9. Incubate the cell suspension on ice for 15 min. And quickly aliquot 50μL (or 100μL) of the cells into pre-cooled microcentrifuge tubes.
10. Immediately freeze the cells in a dry ice/isopropanol bath or liquid nitrogen, and store at -80°C.
Note
1. The growiing phase (O.D.600) of 50 mL culture is critically important to the quality of competent cells.
2. From the step 4 on, keep every process on ice and do it quickly. High temperature and long processing time will decrease the cell quality.
3. For saving time, ask somebody to help you opening, recapping and freezing the microcentrifuge tube while aliquoting the cell suspensions.
4. Test your competent cell efficiency (c.f.u.) with 1 pg of plamid DNA by conventional Heat-Shock transformation. The effeciency of a good-quality competent cell should be higher than 107 c.f.u.
