Materials Required
- Elution buffer
- Scalpel blade
- UV transilluminator
- Agarose
- Micro pipettes
- Micro pipette tips
- Dry bath incubator
- Microfuge tubes
- Centrifuge
- N-Butanol
- Cryo box
- Cyclomixer
- 70% Ethanol
- 95% Ethanol
- TE buffer
- -20oC freezer
- -70oC freezer
Procedure
1. Visualize the low melting point agarose gel with DNA bands under a UV transilluminator and locate the desired DNA band to cut.
2. Carefully cut around the desired DNA band using a scalpel blade.
3. Transfer the gel piece into a microfuge tube.
4. Add elution buffer into the microfuge tube until the level of buffer is just above the level of gel slice.
5. Heat the gel slice at 65oC until it melts.
6. Freeze the melted gel with DNA by placing in a -70oC freezer for10minuts.
7. After freezing, centrifuge for 10minutes and transfer the supernatant into a new microfuge tube.
8. Again add half amount of elution buffer that you added in the previous step into the pellet.
9. Heat at 65oC until the agarose melts.
10. Freeze the melted gel with DNA by placing in a -70oC freezer for10minuts.
11. Centrifuge the tube again for 10 minutes and transfer (pool) the supernatant into the previous tube with supernatant.
12. Discard the tube with pellet.
13. Add an equal volume of n-Butanol to the supernatant and mix the contents well.
14. Vortex the tube for 15 minutes in order to remove the Ethidium bromide.
15. Discard the upper phase of butanol and repeat the process by adding n-butanol again for one or more times.
16. Add 2 times volume of 95% ethanol and mix thoroughly.
17. Keep for precipitation in -70oC freezer for 30minutes to overnight.
18. After precipitation, centrifuge for 15 minutes.
19. Discard the supernatant into a waste beaker and add 200µl of 70% ethanol to the pellet.
20. Centrifuge for 5minutes and discard the supernatant again.
21. Allow the pellets to dry well.
22. Suspend the pellets in 20µl of TE buffer. (If you want to confirm the recovered DNA, run (1µl) it on a gel.
23. The recovered DNA can be now used for further process of cloning otherwise can stored in -20oC freezer.
Help:
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