Genomic DNA Extraction
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Bacterial Genomic DNA Extraction
Protocol
- Culture 5 ml of the bacteria strain in LB medium until stationary phase (overnight)
- Centrifuge 2 ml of culture 10 min at room temperature
- Resuspend the pellet in 540 µl of T10E1 + 30 µl of 10% SDS + 30 µl pronase 20mg/ml
- Incubate 1h at 37˚C
- Add 125 µl 4M NaCl and 80 µl of CTAB/NaCl solution
- Incubate 10 min at 65˚C
- Extract with same volume of Chloroform/isoamyl alcohol (24:1)
- Centrifuge 5 min at RT
- Take the upper phase in a fresh tube and extract with same volume of phenol/( Chloroform/isoamyl alcohol) (1:1)
- Centrifuge 5 min at RT
- Add 0.6 volume of isopropanol to the upper phase
- Leave at -80˚C for 10 min
- Centrifuge 20 min at 4˚C
- Wash the pellet with 100 µl of 70% ethanol
- Dry and resuspend in 100 µl of T10E1 and 1µl RNAse
Notes
- (should obtain 5-7 µg/µl)
- pronase=protease from Streptomyces griseus
- T10E1: TrisHCl pH7.0 10 mM, EDTA 1mM
- CTAB/NaCl solution: 4.1 g NaCl+ 10 g CTAB in 80 ml H2O
- Stir and heat at 65˚C
- Adjust to 100 ml (viscous)