Protocol: Calcium Chloride treated bacterial cell transformation

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Tony Rook
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Protocol: Calcium Chloride treated bacterial cell transformation

Please find the link and protocol for the following method:

Calcium Chloride treated bacterial cell transformation

http://www.genome.ou.edu/protocol_book/protocol_partII.html#II.F

A brief background discussion of transformation and transfection can be found in the Appendix.

For DNA transformation (14,15), the entire DNA ligation reaction is added to an aliquot of competent cells, which is mixed gently, and incubated in an ice-water bath. This mixture then is heat-shocked briefly in a 42degC water bath for 2-5 minutes. At this point in the transformation, the method varied slightly depending on whether the cloning vector is M13-based or pUC-based.

For M13-based transformation (14), an aliquot of non-competent cells is added to the heat-shocked mixture, as is the lac operon inducer homologue, IPTG, and the b-galactosidase chromogenic substrate, x-gal. Melted top agar is added, and the transformation mixture then is poured onto the surface of an agar plate. After the top agar solidified, the plates are inverted and incubated overnight at 37degC.

For pUC-based transformation (15), an aliquot of liquid media is added to the heat-shocked mixture, which then is incubated in a 37degC water bath for 15-20 minutes. After recovery, the cell suspension is concentrated by centrifugation and then gently resuspended in a smaller volume of fresh liquid media. IPTG and x-gal are added to the cell mixture, which is spread onto the surface of an ampicillin-containing agar plate. After the cell mixture had diffused into the agar medium, the plates are inverted and incubated overnight at 37degC.

Protocol

1. Add the entire ligation reaction to a 12 X 75 Falcon tube containing 0.2-0.3 ml of competent cells, mix gently, and incubate in an ice-water bath for 40-60 minutes. (For retransformation of recombinant DNA, add approximately 10-100 ng of DNA directly to competent cells).

2. Heat shock the cells by incubation at 42degC for 2-5 minutes.

For M13-based transformation:

3a. Add the following reagents to the heat shocked transformation mixture:

Non-competent cells 0.2 ml
IPTG (25 mg/ml H2O) 25 ul
x-gal (20 ml/ml DMF) 25 ul
lambda top agar 2.5 ml

4a. Mix by briefly vortexing, and then quickly pour onto the surface of a pre-warmed lambda agar plate.

5a. Allow 10-20 minutes for the agar to harden, and then invert and incubate overnight at 37degC.

For pUC-based transformation:

3b. Add the following reagents to the heat shocked transformation mixture, add 1 ml of fresh 2xTY and incubate in a 37degC water bath for 15-30 minutes.

4b. Collect the cells by centrifugation at 3000 rpm for 5 minutes, decant the supernatant, and gently resuspend in 0.2 ml of fresh 2xTY.

5b. Add 25 ul IPTG (25 mg/ml water) and 25 ul x-gal (20 mg/ml DMF), mix and pour onto the surface of a pre-warmed LB-Amp plate. Spread over the agar surface using a sterile bent glass rod or sterile inoculating loop.

6b. Allow 10-20 minutes for the liquid to diffuse into the agar, and then invert and incubate overnight at 37degC.

For pBR322, pAT153 or other non-lacZ containing vectors:

3b. Add 1 ml of fresh 2xTY to the cells and incubate for 15-30 minutes at 37 degC. Spread approximately 50 ul on L plates containing antibiotic using a sterile glass spreader. Incubate the plates overnight at 37degC.

Shubhangi
Shubhangi's picture
Hi

Hi
it is very important to calculate transformation efficiency . Or checking how efficient your competent cells are - is very much needed. Simplest formula for this is

Please visit the links that give very basic idea about transformation. Its actually for graduate students but  useful.
http://www.sciencebuddies.org/science-fair-projects/project_ideas/BioChem_p013.shtml
http://www.tracy.k12.ca.us/thsadvbio/pdfs/transefficiency.pdf