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| Forward primer Ps-1 | 5′- agt cga acg gca gcg ggg g -3′ |
| Reverse primer Ps-2 | 5′- ggg gat ttc aca tcg gtc ttg ca -3′ |
Expected amplicon size from R. solanacearum template DNA = 553 bp.
| a Methods were validated using Taq polymerase from Perkin Elmer (AmpliTaq) and Gibco BRL. | ||
| N.B. Originally optimised for MJ Research PTC 200 thermocycler with Gibco Taq Polymerase. Perkin Elmer AmpliTaq and buffer can also be used at the same concentrations. | ||
| Reagent | Quantity per reaction | Final concentration |
|---|---|---|
| Sterile UPW | 16,025 µl | |
| 10X PCR buffer a | 2,5 µl | 1X (1,5 mM MgCl 2 ) |
| BSA (fraction V) (10 %) | 0,25 µl | 0,1 % |
| d-nTP mix (20 mM) | 0,125 µl | 0,1 mM |
| Primer Ps-1 (10 µM) | 0,5 µl | 0,2 µM |
| Primer Ps-2 (10 µM) | 0,5 µl | 0,2 µM |
| Taq polymerase (5U/µl) a | 0,1 µl | 0,5 U |
| Sample volume | 5,0 µl | |
| Total volume: | 25,0 µl | |
Run the following programme:
| 1 cycle of: | (i) | 5 minutes at 95 °C (denaturation of template DNA) |
| 35 cycles of: | (ii) | 30 seconds at 95 °C (denaturation of template DNA) |
| (iii) | 30 seconds at 68 °C (annealing of primers) | |
| (iv) | 45 seconds at 72 °C (extension of copy) | |
| 1 cycle of: | (v) | 5 minutes at 72 °C (final extension) |
| (vi) | hold at 4 °C. |
NB: This programme is optimised for use with an MJ Research PTC 200 thermal cycler. Modification of the duration steps of cycles (ii), (iii) and (iv) may be required for use with other models. U.K.
PCR products amplified from R. solanacearum DNA produce a distinctive restriction fragment length polymorphism with enzyme Taq I after incubation at 65 °C for 30 minutes. The restriction fragments obtained from R. solanacearum -specific fragment are 457 bp and 96 bp in size.]