DNA extraction from E. coli:
Materials required:
- Overnight culture of coli in LB
- TE buffer ( 10mM tris.Cl, 1mM EDTA, pH 8.0)
- 10% SDS
- 1:1 Phenol-Chloroform mixture
- Chloroform
- 5N NaCl
- 5M Ammoniun acetate
- Ice cold isopropanol
- 70% ethanol
Protocol of DNA extraction from E.coli:
- Take 1.5 ml of bacterial broth culture (overnight culture of coli in LB) into a microfuge tube.
- Centrifuge at 800rpm for 10 minutes at 4°C and discard the supernatant.
- Suspend the pellet in 400µl TE buffer. Mix well by vortexing.
- Add 10µl of 10% SDS and mix it.
- Incubate the tube at 37°C for 1 hour in water bath.
- Shear the cell suspension 3-5 times with the help of 26G needle.
- Add 500 µlof 1:1 phenol-chloroform mixture.
- Centrifuge at 13000rpm for 2 minutes at 4°
- Transfer the supernatant into another microfuge tube.
- Add 500 µl of chloroform and centrifuge at 13000rpm for 2 minutes at 4°
- Transfer the supernatant into another microfuge tube.
- Add 25µl of 5N NaCl and centrifuge at 13000rpm for 10 minutes at 4°
- Discard the supernatant and suspend the pellet in 100µl TE buffer.
- Incubate the tube at 37°C for 30 minutes.
- Add 10 µl of 5M ammonium acetate.
- Add 250 µl of cold isopropanol and incubate at room temperature for 5 minutes.
- Centrifuge at 13000rpm for 10 minutes at 4°
- Discard the supernatant and wash the pellet with 100 µl of 70% ethanol.
- Pour off the ethanol and invert the tube on a clean absorbent paper to drain.
- Allow the pellet to air dry for 5-10 minutes.
- Suspend the pellet in 100µl TE buffer.
- Store at -20°