"

From 2010.igem.org

MAIN STEPS/TIMETABLE

Total time required to complete the procedure is 1h 40min.

MATERIALS

- Incubator
- Centrifuge

- Absolute ethanol stored at -20°C
- DNA sample
- 95% ethanol stored at room temperature
- Water

CHECK - LIST PROCEDURE

- Measure out 2 volumes absolute ethanol into DNA sample. (The sample is generally in 1.5mL eppendorf.)
- Incubate at -80°C for 1 hr.
- Centrifuge at a speed of at least 10000 Xg for 30 mins at 0°C, gently aspirate out the supernatant and discard it.
- Measure out 750 - 1000 µl of 95% ethanol into the eppendorf tube that is used at first step.
- Centrifuge at a speed of at least 10000 Xg for 10 mins at 4°C, gently aspirate out the supernatant and discard it.
- Dry the pellet in air until white pellet appears.
- Add appropriate volume of water to pellet.
- Resuspend pellet by vortexing/by shaking vigorously.

NOTES

- Storing the absolute ethanol in -20 is recommended.
- Long incubation time is critical for small fragments.
- Another critical step for small fragments under 200 base pairs is the fourth step. Generally, washing involves adding the ethanol and inverting several times.
- The pellet in the 6th step is air dried so that it turns white, showing that all ethanol is eliminated.)
- Many protocols recommend resuspending in 10 mM Tris-HCl or TE. The advantage of TE is that EDTA chelates magnesium ions which makes it more difficult for residual DNases to degrade the DNA. I generally prefer H2O and don't seem to experience problems of this sort. If you plan to ultimately use electroporation to transform your DNA then resuspending in H2O has the advantage of keeping the salt content of your ligation reaction down.
- Incubation in - 80C is done in deep of deep freezer.
- ALL STEPS ARE DONE IN ICE.