Team:Mingdao/Protocol
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Mingdao
PROTOCOLS
PLASMID EXTRACTION
Bacterial culture
For plasmid extraction, we normally cultured E. coli with plasmid DNA in 6 ml LB broth supplemented with chloramphenicol (34 μg/ml) or ampicillin (100 μg/ml) at 37°C overnight.
Plasmid Extraction Procedure
In all of our experiments, we used a mini-prep kit
(Presto™ Mini Plasmid Kit) developed by GENEAID BIOTECH LTD., Taiwan. And we followed the manufacturer's instructions for plasmid extraction. Below is the brief protocol provided by Geneaid Biotech Ltd., Taiwan. And you can also go to their website for more information and complete protocol.
PCR FOR GENE CLONING
We always used a high-fidelity DNA polymerase named KOD -Plus- provided by TOYOBO CO., LTD. We followed the manufacturer's instructions. Conditions and reagents were briefly shown below.
PCR CHECK – COLONY PCR
To quick check the gene insertion onto the vector, we performed colony PCR for screening single colonies on agar plates. We used a kit of Taq polymerase (JMR Holdings) and followed the manufacturer's instructions. Brief conditions were shown below.
DNA GEL ELECTROPHORESIS
We prepared 1% DNA agarose gels and run gel with 6X FluoroDye DNA Loading Dye (Green) and FluoroBand 1KB (0.25-10kb) Fluorescent DNA Ladder. The images were pictured with Omega Lum™ G Imaging System.
PCR CLEANUP & GEL ELUTION
We used GenepHlow™ Gel/PCR Kit developed by GENEAID BIOTECH LTD., Taiwan and followed the manufacturer's instructions.
RESTRICTION ENZYME
EcoRI, XbaI, SpeI, PstI and others were used in this study and purchased from New England Biolabs (NEB). We have three conditions as described below used for plasmid check and gene cloning, respectively.
Plasmid check
Gene cloning (for vectors)
Gene cloning (for inserts)
TRANSFORMATION – HEAT SHOCK
We used and purchased Elite Competent Cells (DH5α) from GENEAID BIOTECH LTD. We followed the provided protocol for transformation procedure and briefly described below.
↓ Thaw one tube of competent cells on ice from -80°C refrigerator
↓ Add 5 µl of DNA into 50 µl of competent cells. Operate in a laminar flow and manipulate DNA and competent cells on ice.
↓ Vortex for 1 sec and stay on ice for 5 min
↓ Heat-shock the cells at 42°C for 1 min
↓ Incubate on ice for 5 min
↓ Recover with 500 µl of LB media and shake in an incubator at 37°C for 1 hr. Operate in the laminar flow.
↓Spread the cells onto a LB agar plate supplemented with 34 µg/ml of chloramphenicol or 100 µg/ml of ampicillin. Operate in the laminar flow.
↓ Thaw one tube of competent cells on ice from -80°C refrigerator
↓ Add 5 µl of DNA into 50 µl of competent cells. Operate in a laminar flow and manipulate DNA and competent cells on ice.
↓ Vortex for 1 sec and stay on ice for 5 min
↓ Heat-shock the cells at 42°C for 1 min
↓ Incubate on ice for 5 min
↓ Recover with 500 µl of LB media and shake in an incubator at 37°C for 1 hr. Operate in the laminar flow.
↓Spread the cells onto a LB agar plate supplemented with 34 µg/ml of chloramphenicol or 100 µg/ml of ampicillin. Operate in the laminar flow.
TRANSFORMATION – ELECTROPORATION
Electroporation is a transformation method that relies on providing an electric current through the cell and creating holes or pores in the cell membrane. Through those pores, the plasmid will enter the cell before they are getting closed.
To do this transformation, the equipment and materials needed are listed below:
1. Electrocompetent cells
2. Competency medium
3. Plasmid you want to transform
4. Electroporator
5. Electroporation cuvettes
To do this transformation, the equipment and materials needed are listed below:
1. Electrocompetent cells
2. Competency medium
3. Plasmid you want to transform
4. Electroporator
5. Electroporation cuvettes
E. coli Nissle
Preparation of electrocompetent cells
↓Culture E. coli Nissle in LB broth at 37°C overnight
↓Make 1 ml aliquots in each eppendorf
↓Centrifuge the bacteria at 15,000 x g for 2 min at 4°C
↓Repeat wash with 1 ml of sterile H2O and centrifuge at 15,000 x g for 2 min at 4°C until the pellets are unable to stick tightly on the wall of eppendorf.
(If the pellets begin to slip out the wall, then centrifuge the last time at 16,000 x g for 6 min)
↓Make 1 ml aliquots in each eppendorf
↓Centrifuge the bacteria at 15,000 x g for 2 min at 4°C
↓Repeat wash with 1 ml of sterile H2O and centrifuge at 15,000 x g for 2 min at 4°C until the pellets are unable to stick tightly on the wall of eppendorf.
(If the pellets begin to slip out the wall, then centrifuge the last time at 16,000 x g for 6 min)
Electroporation with the BTX™ Gemini X2 Electroporation System
↓Transfer 40 µl of the electrocompetent cells to the 2-mm-gap electroporation cuvette
↓Perform electroporation at 2,500 Volt, 25 µF, 200Ω with a single electric pulse of 4.5 ms.
↓Flush the electroporated mixture out of the cuvette with 360 µl of water
↓Add equivalent double strength LB (two-fold concentration)
↓Recover the cells at 37°C for 1 hr
↓Spread 50-100 µl of bacteria on LB agar plate with 20 µg/ml of chloramphenicol
↓The next day, check the colony by PCR.
↓Perform electroporation at 2,500 Volt, 25 µF, 200Ω with a single electric pulse of 4.5 ms.
↓Flush the electroporated mixture out of the cuvette with 360 µl of water
↓Add equivalent double strength LB (two-fold concentration)
↓Recover the cells at 37°C for 1 hr
↓Spread 50-100 µl of bacteria on LB agar plate with 20 µg/ml of chloramphenicol
↓The next day, check the colony by PCR.
S. mutans
Preparation of electrocompetent cells
↓Cultivate S. mutans in BHI overnight at OD600 around 0.6
↓Vortex the bacteria gently and make 1.5 ml aliquots in each eppendorf
↓Centrifuge the bacteria at 4000 x g for 15 min at 4°C
↓Wash twice with ice-cold buffer (10mM HEPES at a pH of 7.0, 15% glycerol)
↓Resuspend in the electroporation buffer (5% sucrose, 15% glycerol)
↓Vortex the bacteria gently and make 1.5 ml aliquots in each eppendorf
↓Centrifuge the bacteria at 4000 x g for 15 min at 4°C
↓Wash twice with ice-cold buffer (10mM HEPES at a pH of 7.0, 15% glycerol)
↓Resuspend in the electroporation buffer (5% sucrose, 15% glycerol)
Electroporation with the BTX™ Gemini X2 Electroporation System
↓40μl of ice-cold electro-competent cells in a 1-mm gap cuvette
↓Electroporation by a single electric pulse of 4.5 ms with a setting of 1,250 V, 25 µF, 200Ω)
↓Immediately add fresh 960 µl of BHI broth, and incubate at 37°C for 1 hr
↓Plate the cells onto BHI agar plate supplemented with 1 mg/ml of spectinomycin
↓Grow cells for 2 days at 37°C anaerobically
↓Check colonies by PCR
↓Electroporation by a single electric pulse of 4.5 ms with a setting of 1,250 V, 25 µF, 200Ω)
↓Immediately add fresh 960 µl of BHI broth, and incubate at 37°C for 1 hr
↓Plate the cells onto BHI agar plate supplemented with 1 mg/ml of spectinomycin
↓Grow cells for 2 days at 37°C anaerobically
↓Check colonies by PCR