IGEM:University of Debrecen:PCR Purification from Solution

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PCR products purification from Solution:

Scientific Background

Overview

Materials

Centrifuge

Vortex

pipett (100-1000µl and 2-20µl)

tips (blue and yellow)

ice and ice container

Trash

PCR product

Binding Buffer

Wash Buffer

Elution Buffer

High Pure Micro Filter Tubes

Collection Tubes

Procedure

1. PCR products purification from Solution:

2. 100µl of the PCR product

3. Add 400µl Binding Buffer

4. Vortex it two times for 4 sec. each

5. Centrifuge the mixture briefly

6. Insert one H.P.F.T. into one Collection Tube

7. Transfer the sample from step 1 to the upper reservoir of the Filter Tube

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

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

10. Reconnect the Filter Tube to the same Collection Tube

11. Add 400 µl Wash Buffer

12. Centrifuge 30-60 sec at 8000 x g C

13. Discard the followthrough solution

14. Reconnect the Filter Tube to the same Collection Tube

15. Add 300µl Wash Buffer

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

17. Discard the followthrough solution

18. Reconnect the Filter Tube to the same Collection Tube

19. Centrifuge 1 min at maximum speed

20. Discard the followthrough solution and the Collection Tube

21. Connect the filter Tube to a clean 1.5 ml eppendorf tube

22. Add 10-20 µl Elution Buffer to the center of the Filter Tube

23. Centrifuge 1 min at 8000 x g

Notes & troubleshooting

References

1 Vogelstein, B. et al. (1979) Preparative and analytical purification of DNA from agarose 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 Diabetes 51, 2982-2988.

3 Chang, PC et al. (2001) Complete nucleotide sequence of avian paramyxovirus type 6 isolated from ducks J. Gen. Virol. 82, 2157-2168.

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