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Competent Cells R BCL

This document describes a protocol for making E. coli cells competent for transformation using the RbCl method. It details the preparation of media and solutions including YETM, TFB1, and TFB2. The protocol then outlines the steps to prepare competent cells including growing cultures, chilling, washing cells with TFB buffers, and freezing aliquots. Instructions are provided for transforming the competent cells with plasmid DNA and plating to select colonies. Finally, a method is described for testing the transformation efficiency of the competent cells using pUC19 plasmid DNA.

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62 views2 pages

Competent Cells R BCL

This document describes a protocol for making E. coli cells competent for transformation using the RbCl method. It details the preparation of media and solutions including YETM, TFB1, and TFB2. The protocol then outlines the steps to prepare competent cells including growing cultures, chilling, washing cells with TFB buffers, and freezing aliquots. Instructions are provided for transforming the competent cells with plasmid DNA and plating to select colonies. Finally, a method is described for testing the transformation efficiency of the competent cells using pUC19 plasmid DNA.

Uploaded by

me_dayakar
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
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Transformation Protocol: RbCl-method

I. Media and Solutions


YETM , 500 mls: 2.5 g Yeast Extract
10 g Tryptone
5g MgSO4 •7H2 O
pH to 7.5 with KOH For plates: add 7.5 g of Agar

TFB1, 200 mls: 0.59 g KOAc (30 mM)


2.42 g RbCl (100 mM)
0.29 g CaCl2 •2H2 O (10 mM)
1.98 g MnCl 2 •4H2 O (50 mM)
30 ml Glycerol (15%)

A. Adjust to pH 5.8 with 0.2 M acetic acid (do not adjust pH with KOH).
B. Add ddH2 O to 200 ml. Filter sterilize. Store refrigerated at 4°C

TFB2, 200 mls: 0.42 g MOPS (10 mM)


2.21 g CaCl2 - 2 H 2 O (75 mM)
0.24 g RbCl (10 mM)
30 ml Glycerol (15%)

A. Adjust to pH 6.5 with KOH.


B. Add ddH2 O to 200 ml. Filter sterilize. Store refrigerated at 4°C

II. Preparation of Competent Cells


1. Plate cells from frozen stock onto YETM plate and incubate overnight (O/N) at 37°C. Do not omit
this step. Do not use colony or cells stored at 4°C to inoculate O/N culture in step 2.

2. Pick a single fresh colony the next day, inoculate 5 mls of YETM medium and grow O/N at 37°C.

• DO NOT vortex cells at any time after this point in the procedure.

3. Dilute 1 ml of O/N culture into 50 mls YETM medium prewarmed to 37°C, and grow at 37°C for 2h
with agitation. Volumes can be scaled up 5X and all of the 5 ml O/N culture can be used.

4. Chill 5 minutes on ice and spin in IEC centrifuge for 10 minutes at 2000 rpm at 4°C. Prechill the
centrifuge to 4°C. Prechill centrifuge tubes on ice.

• DO NOT allow the cells warm up over 4°C at any time in the procedure.

5. Immediately aspirate off all of the supernatant. Resuspend the cells in 10 ml of ice-cold TFB1 with
gentle re-pipetting. Use chilled pipette(s).

6. Incubate cells for 5 minutes on ice. Spin in IEC centrifuge for 10 minutes at 2000 rpm at 4°C.

7. Immediately aspirate off all of the supernatant. Resuspend the cells in 2.0 ml of ice-cold TFB2
with gentle re-pipetting. Use chilled pipette or pipette tip(s).

8. Incubate cells on ice for 15 minutes. Cells may be used for transformation now (next page), or
frozen as described below.

To freeze: aliquot cells in 220 µl volumes into prechilled 0.5 ml microfuge tubes (on ice). Freeze
immediately on dry ice. Store cells frozen at -80°C.

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III. Transformation of Competent Cells

1. Thaw cells at room temperature until just thawed and then place on ice for 10 minutes.

2. Add to 15 ml plastic round bottom tube on ice: 0-9 µl TE


1-10 µl DNA (10-100 ng)
10 µl final volume

• In general, the maximum amount of DNA that should be used is 0.1 volume of the cells. Add no
more than 100 ng of DNA.

3. Add 100 µl of competent cells and mix by gentle repipetting.

4. Incubate cells on ice for 20-30 minutes.

5. Heat shock the cells exactly 90 seconds at 42°C.

6. Return cells on ice for 2 minutes.

7. Add 1 ml of YETM medium and incubate at 37°C for 45 minutes to 1 hour, with slow gentle shaking.

• For blue/white color selection, spread IPTG and X-gal on plates now. Place at 37°C until use.

8. Plate 0.1 - 0.2 ml of the transformed cells on LB-plates containing the appropriate antibiotic,
with IPTG and X-gal if needed. Incubate overnight at 37°C. Place at 4°C to store and/or enhance
blue color.

IV. Testing Competent Cells

1. Transform 100 µl of cells with 1 µl (10 pg) of pUC19 monomer (0.01 µg/µl).

2. Plate 0.25 ml of transformation mixture. Incubate overnight at 37°C.

3. Efficiency = # of colonies X 4 X 105 = # of colonies per µg. Should obtain 1-5 X 107 /µg after one
freeze-thaw cycle.

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