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o 1.4 Note
Glycerol 15%(w/v)
• RF2 buffer
Glycerol 15%(w/v)
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 (For DH5α)
should be higher than 107 c.f.u.
Please find the link and protocol for the following method:
There are two main methods for preparation of competent bacterial cells (14) for transformation,
the calcium chloride and the electroporation method. For the calcium chloride method, a glycerol
cell culture stock of the respective E. coli strain is thawed and added to 50 ml of liquid media.
This culture then is preincubated at 37degC for 1 hour, transferred to an incubator-shaker, and is
incubated further for 2-3 hours. The cells are pelleted by centrifugation, resuspended in calcium
chloride solution, and incubated in an ice-water bath. After another centrifugation step, the
resulting cell pellet again is resuspended in calcium chloride to yield the final competent cell
suspension. Competent cells are stored at 4degC, for up to several days.
1. Thaw a frozen glycerol stock of the appropriate strain of E. coli, add it to an Erlenmeyer flask
containing 50 ml of pre-warmed 2xTY (1) media, and pre-incubate in a 37degC water bath for 1
hour with no shaking. Further incubate for 2-3 hours at 37degC with shaking at 250 rpm.
2. Transfer 40 ml of the cells to a sterile 50 ml polypropylene centrifuge tube, and collect the
cells by centrifugation at 3000 rpm for 8 minutes at 4deg C in a GPR centrifuge (Beckman) or
6000 rpm for 8 minutes at 4degC in an RC5-B centrifuge (DuPont) equipped with an SS-34
rotor. For M13-based transformation, save the remaining 10 ml of culture in an ice-water bath
for later use.
3. After centrifugation, decant the supernatant and resuspend the cell pellet in one-half volume
(20 ml) of cold, sterile 50 mM calcium chloride, incubate in an ice-water bath for 20 minutes,
and centrifuge as before.
4. Decant the supernatant and gently resuspend the cell pellet in one-tenth volume (4 ml) of cold,
sterile 50 mM calcium chloride to yield the final competent cell suspension.
Electroporation Protocol
Preparation of Electro-competent Cells:
Reference:
Rakesh C. Sharma and Robert T. Schimke, "Preparation of Electro-competent E. coli Using Salt-
free Growth Medium", Biotechniques 20, 42-44 (1996).
5. Replies
Posted By omehenk
on 5/2/2007 11:39 AM
Adjust to pH 5.8 with 0.2 M acetic acid (do not adjust pH with KOH).
Add ddH2O to 200 ml. Filter sterilize. Store refrigerated at 4°C
T F B 2 , 200 mls:
0.42 g MOPS (10 mM)
2.21 g CaCl2-2H2O (75 mM)
0.24 g RbCl (10 mM)
30 ml Glycerol (15%)
Adjust to pH 6.5 with KOH.
Add ddH2O to 200 ml. Filter sterilize. Store refrigerated at 4°C
II. Preparation of Competent Cells
Pick a fresh colony or scrape some from a glycerol stock and grow up an
overnight culture in LB, shake at 37C.
Dilute overnight culture 1 in 100 into 200mL of LB (pre-warmed to 37C).
Grow at 37C (shaken) for 2-3 h, until OD600 0.25-0.3.
Incubate on ice for 5 min.
Spin at 3000g, 5 min 4C. Discard supernatant.
Resuspend pellet in 40 ml Tfb I for a 200 ml prep. Do NOT Vortex. Pipet
gently.
Incubate on ice, 5 min.
Centrifuge at 3000g, 5 min 4C. Discard supernatant.
Resuspend pellet in 4 ml Tfb II. Be extremely gentle. Do not pipet cells!
Incubate on ice, 15 min.
Make aliquots of 100 ul. Freeze on dry ice. Store at -80C.
6.