Transformation of drug-resistant plasmid DNA Protocol

Experiment Summary

The Ca treatment of the recipient bacterium E.coli RRI at low temperature changes the permeability of the cell membrane and facilitates the uptake of DNA by the recipient bacterium, and this Ca-treated bacterium is called the receptor state. pBR322 plasmid carries the genes of aminobenzyl penicillin (Ap) and tetracycline (Tc), and if the pBR322 plasmid is transferred into the E.coli RRI bacterium, which is sensitive to the above antibiotics, it can cause this bacterium to acquire Apr and Tcr and exhibit resistance to Ap and Tc.

Main Reagents

1. LB liquid medium

2. 75mM CaCl₂

3. Ampicillin agar plate (50 ug/ml)

Main Equipment

1. Sterilized small test tubes

2. Graduated pipette

3. Microvolume sparger

4. Centrifuge

5. L-shaped glass rod

Experimental Materials

1. Strain E.coli RRI

2. pBR322 plasmid DNA

3. 75 mmol CaCl₂ for the receptor bacteria

Experimental Steps

1. Preparation of receptor bacteria

1) Inoculate the bacteria in 5 ml LB liquid medium and incubate at 37℃ for 16-20 h with shaking.

2) Take fresh bacterial solution, inoculate in LB medium at 1/100 inoculum, and incubate at 37℃ for 2-3 h with shaking.

3) Take 1.5 ml of bacterial broth, centrifuge at 4℃ 3000 rpm/min for 5 min, discard supernatant.

4) Add 750 μl of pre-cooled (4℃) 75 mmol/L CaCl₂ solution to the bacterial precipitate, gently blow the bacterial suspension and put it in ice bath for 30 min.

5) Centrifuge at 3000 rpm/min at 4℃ for 5 min, discard the supernatant.

6) Add 200 μl of pre-chilled 75 mmol/L CaCl₂ solution to the bacterial precipitate, put it in ice bath for more than 4 min. 4℃ for storage.

2. Plasmid transformation

1) Take 2 clean, sterilized EP tubes and add each component.　

2) Mix the above 2 tubes well and put them in ice bath for 30 min.

3) Place in ice bath for 2 min at 42℃ or 5min at 37℃.

4) Add 1 ml of LB broth medium and incubate for 1 h at 37℃.

3. Transformer screening

1) Take the culture solution of the experimental group and apply it on ampicillin agar plate (50 ug/ml), and the control group's solution was applied on drug-free LB agar plate and ampicillin agar plate respectively, each plate was coated with 0.1 ml and well coated with sterilized L-shaped glass rod. The coated plates were incubated at 37℃ overnight to observe the transformation results.

2) E.coli recipient bacteria do not contain plasmid DNA and do not contain Apr and Tcr. If the experimental group cannot grow in the medium containing Ap and Tc, if the colonies appear on the plates containing Ap and Tc, it can be initially determined that the recipient bacteria have obtained the drug-resistant plasmid, and then further identify the transformants by extracting the plasmid DNA.

* For research use only. Not intended for any clinical use.

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Transformation of drug-resistant plasmid DNA Protocol

Experiment Summary

Main Reagents

Main Equipment

Experimental Materials

Experimental Steps

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