Team:Debrecen-Hungary/protocols/PCR purification from Agarose Gel

DNA Fragments Purification from Agarose Gel

Scientific Background

DNA fragments migrate with different speed in an agarose gel during electrophoresis. As a result different fragments can be separated and isolated by cutting them out of the gel and purification of the DNA from the gel.

There are several ways how this procedure can be performed. As a general rule take into consideration that UV light is dangerous for DNA, so expose your gel for the minimum time to UV light.

Alternatives are the dyes that give a fluorescent signal without UV exposure. We used Gel Red and Blue Box, but had serious problems, because gel red gave sometimes strange smears of the DNA bands.

Once you cut out the agarose with the apropriate fragment size, there are several options to melt it. E.g agarase digestion, or special kits. We used Roche PCR purification Kits with excellent results.

Overview

You will need weight to measure the gel fragment.

The whole procedure should be not longer than 90 minutes for two fragment, including incubation times.

Materials

Centrifuge

Vortex

Incubator

Pipet (100-1000µl and 2-20µl)

Tips (blue and yellow)

Ice and ice container

Trash

DNA fragments in Agarose Gel

Binding Buffer

Binding Enhancer

Wash Buffer

Elution Buffer

High Pure Micro Filer Tubes

Collection Tubes

scale

Procedure

1. Identify bands by staining gel with ethidium bromide or SYBR Green I

2. Cut desired DNA band from gel using a scalpel or razor blade that has been sterilized with ethanol

3. Preweight an empty, sterile 1.5 ml eppendorf tube

4. Place excised agarose gel slice in the sterile eppendorf tube

5. determine gel weight by reweighing the tube containing the gel slice and subtracting the weight of the empty tube

6. Add 300µl Binding Buffer for every 100mg agarose gel in the tube

7. Vortex 15-30 sec

8. Incubate the suspension for 10 min at 56 C

9. Vortex every 2-3 min during incubation

10. Add 100µl Binding Enhancer for every 10 mg agarose gel slice in the tube

11. Vortex thoroughly

12. Centrifuge the mixture briefly

13. Insert one High Pure Filter Tube into one Collection Tube

14. Transfer the sample from step 12 to the upper reservoir of the Filter Tube

15. Centrifuge 30-60 sec at 8000 x g at 15-25 C

16. Disconnect the Filter Tube, and discard the followthrough solution

17. Reconnect the Filter Tube to the same Collection Tube

18. Add 400 µl Wash Buffer

19. Centrifuge 30-60 sec at 8000 x g at 15-25 C

20. Discard the followthrough solution

21. Reconnect the Filter Tube to the same Collection Tube

22. Add 300 µl Wash Buffer

23. Centrifuge 30-60 sec at 8000 x g at 15-25 C

24. Discard the followthrough solution

25. Reconnect the Filter Tube to the same Collection Tube

26. Centrifuge 1 min at maximum speed

27. Discard the followthrough solution

28. Connect the Filter Tube to a clean 1.5 ml eppendorf tube

29. Add 10-20 µl Elution Buffer to the centre of the Filter Tube

30. Centrifuge 1 min at 8000 x g

Notes & troubleshooting

References

1 Vogelstein, B. et al. (1979) Preparative and analytical purification of DNA from agarose[http://www.pnas.org/content/76/2/615.full.pdf+html Proc Natl Acad Sci USA 76 (2):615-619].

2 Löbner, K. et al. (2002) Different Regulated Expression of the Tyrosine Phosphatase-Like Proteins IA-2 and Phogrin by Glucose and Insulin in Pancreatic Islets [http://diabetes.diabetesjournals.org/content/51/10/2982.full Diabetes 51, 2982-2988].

3 Chang, PC et al. (2001) Complete nucleotide sequence of avian paramyxovirus type 6 isolated from ducks [http://vir.sgmjournals.org/cgi/content/full/82/9/2157 J. Gen. Virol. 82, 2157-2168].

PCR

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