Team:ICT-Mumbai/Protocols
Materials
1. Overnight E. coli culture
2. 10 ml LB medium in culture tubes
3. 0.1 M CaCl2
4. 0.1 M CaCl2 containing 25% glycerol
Equipment
1. Spectrophotometer or plate reader
2. Centrifuge
Procedure
1. Subculture 100 µl overnight grown culture of E. coli in 10 ml LB medium. Shake at 200 rpm at 37°C.
2. After ~2 hours, using either a spectrophotometer or a plate reader, measure absorbance of the culture at 600 nm. At OD600 = 0.2, harvest the cells by centrifugation.
3. Wash cells with ice cold solution of 0.1 M CaCl2 two to three times.
4. Resuspend cells in 100 μl of 0.1 M CaCl2 containing 25% glycerol and store at –80°C till further use.
Materials
1. E. coli competent cells
2. LB medium
3. DNA (plasmid or ligation mix) to be transformed
4. Ice
Equipment
1. Water bath set at 42°C
Procedure
1. Remove the tube containing 100 μl of frozen competent E. coli cells from –80°C and place on ice and allow it to thaw.
2. Add 100–200 ng of DNA to 100 μl of competent E. coli cells. Gently mix by tapping the tube several times.
3. Set up transformation with a positive control and negative control along with transformation mix.
4. Incubate on ice for 20 minutes.
5. After completion of 20 minutes, heat shock the cells for 90 seconds in a water bath set at 42°C.
6. After heat shock, immediately keep the cells on ice for 5 minutes.
7. Remove the tube from ice and add 1 ml of LB medium and incubate at 37°C for 1 hour at 200 rpm.
8. Plate 100-200 μl of transformation mix on LB plates containing appropriate antibiotic(s).
9. Incubate the plates overnight at 37°C.
Materials
1. Overnight E. coli culture
2. 10 ml LB medium in culture tubes
3. 10% glycerol
Equipment
1. Spectrophotometer or plate reader
2. Centrifuge
Procedure
1. Subculture 100 μl overnight grown culture of E. coli in 10 ml LB medium. Shake at 200 rpm at 37°C.
2. After ~2 hours, using either a spectrophotometer or a plate reader, measure absorbance of the culture at 600 nm. At OD600 = 0.2, harvest the cells by centrifugation.
3. Wash cells two to three times with ice cold 10% glycerol.
4. Resuspend cells in 100 μl 10% glycerol and proceed for electroporation.
Materials
1. E. coli electrocompetent cells
2. LB medium
3. DNA to be transformed
4. Ice
5. Bio-Rad Gene Pulser®/MicroPulser™ Electroporation Cuvettes, 0.1 cm gap (#1652089)
Equipment
1. Bio-Rad GenePulser Xcell™ electroporator
Procedure
1. Add 200 ng of DNA to 100 μl of electrocompetent E. coli cells to a chilled microfuge tube placed on ice. Gently mix by tapping the tube several times.
2. Set up transformation with a positive control and negative control along with transformation mix.
3. Transfer the contents of the tube to chilled cuvettes for electroporation.
4. Electroporate using the following conditions:
Voltage (kV): 1.8
Capacitance (µF): 25
Resistance (Ω): 200
Cuvette (mm): 1
5. Immediately after electroporation add 1 ml LB medium to the cuvette, gently mix by inverting and transfer contents to a microfuge tube.
6. Incubate at 37°C for 1 hour at 200 rpm.
7. Plate 100-200 μl of transformation mix onto LB plates containing appropriate antibiotics.
8. Incubate the plates overnight at 37°C.
Materials
1. Overnight culture of E. coli grown in 10 ml LB medium
2. Sterile 1.5 ml microfuge tubes
3. Autoclaved deionized water
4. 100% ethanol
5. PureLink® Genomic DNA Mini Kit, Thermo Fisher Scientific.
Equipment
1. Centrifuge
2. Water bath
Procedure
1. Harvest ~ 1 ml of overnight E. coli cells in a 1.5 ml microfuge tube by centrifugation.
2. Proceed with genomic DNA extraction as per manufacturer’s instructions provided in the kit.
3. Elute in 50–100 μl of autoclaved DNase free water.
4. Store purified gDNA at 4°C for short-term storage and at –20°C for long term storage.
Materials
1. Overnight culture of E. coli grown in 10 ml LB medium
2. Sterile 1.5 ml microfuge tubes
3. Autoclaved sterile water
4. 100% ethanol
5. Exprep Plasmid SV DNA Purification Kit, GeneAll
Equipment
1. Centrifuge
Procedure
1. Harvest 1–5 ml of overnight E. coli cells in a 1.5 ml microfuge tube by centrifugation.
2. Proceed with plasmid DNA extraction as per manufacturer’s instructions provided in the kit.
3. Elute in 50–100 μl of autoclaved DNase-free water.
4. Store purified gDNA at 4°C for short-term storage and at –20°C for long term storage.
PCR clean-up and gel extraction were carried out as per manufacturer’s instructions given in Nucleospin® Gel and PCR Clean-up kit, Macherey-Nagel.