F1ANNEX IITEST SCHEME FOR DIAGNOSIS, DETECTION AND IDENTIFICATION OF RALSTONIA SOLANACEARUM (SMITH) YABUUCHI ET AL.
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Appendix 6Validated PCR protocols and reagents
1.PCR protocol of Seal et al. (1993)
1.2.PCR reaction mix
Reagent | Quantity per reaction | Final concentration |
|---|---|---|
Sterile UPW | 17,65 µl | |
10X PCR buffer34 (15 mM MgCl2) | 2,5 µl | 1X (1,5 mM MgCl2) |
dNTP mix (20 mM) | 0,25 µl | 0,2 mM |
Primer OLI-1 (20 µM) | 1,25 µl | 1µM |
Primer Y-2 (20 µM) | 1,25 µl | 1µM |
Taq polymerase (5U/µl)34 | 0,1 µl | 0,5 U |
Sample volume | 2,0 µl | |
Total volume | 25 µl | |
Method was validated using Taq polymerase from Perkin Elmer (AmpliTaq) and Gibco BRL. | ||
Substituted by Commission Directive 2006/63/CE of 14 July 2006 amending Annexes II to VII to Council Directive 98/57/EC on the control of Ralstonia solanacearum (Smith) Yabuuchi et al..