Biology Protocols

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Medium Scale Rapid Boiling Method

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1. Spin down 1.5ml of an overnight culture of transformed bacteria.

2. Resuspend the pellet in 100µl of STET buffer. Add 5µl of fresh lysozyme solution (optional).

3. Place the tube in a dry heating block at 100oC for 1 minute.

4. Spin the tube for 10 minutes and remove the supernatant to a fresh tube.

5. Add an equal volume of ice-cold isopropanol. Vortex and leave on ice for 5 minutes.

6. Spin down the precipitated nucleic acids for 5 minutes.

7. Resuspend the pellet in 100µl of TE buffer. Add 1/2 volume of phenol. Vortex for 1 minute. Spin for 1 minute.

8. Carefully remove the supernatant to a fresh tube, avoiding the white precipitate at the interface.

9. Add an equal volume of chloroform, vortex and re-spin for 1 minute.

10. Remove the supernatant to a fresh tube. Add 5µl of RNAse A (20mg/ml).

11. Store the sample at -20oC.

 

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