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.
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