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FAIR 3889
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Phytoplasma diseases
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Detection Methods for CMLV
Molecular Methods
RT-PCR assay developed by W. Jelkmann
RT reaction
- Mix the following components in a 1.5 ml microcentrifuge tube:
- 5 µl purified nucleic acid extract
- 1 µl primer mix 100 pmol/µl hexamer and 0.5 µg/µl
oligo-dT17- primer
- 6 µl RNase-free water
- Incubate at 70° C for 5 min. to denature the RNA
- Place on ice and add the following components:
- 4 µl 5 x MMLV RT buffer
- 2 µl 100 mM DTT
- 1 µl 10 mM dNTP mix
- 1 µl MMLV-RT (200 units)
- Incubate at room temperature for 5 minutes
- Incubate at 37° C for 60 minutes
- Incubate at 70° C for 15 minutes
- Store on ice or at -20° C until required for use in PCR
PCR reaction
- 5 µl 10 x buffer
(100 mM Tris-HCl pH 8.3, 750 mM KCl, 35 mM MgCl2)
- 1 µl 10 mM dNTPs
- 1 µl 10 µM primer mix
- 1 µl cDNA
- 1 µl Taq polymerase (approx. 5 units)
- 41 µl sterile water
Primers:
- CML13A: 5´-GCC TGA TCT GCA AAG TGA AG-3´
- CML4A: 5´-TTC GGT CTG AAG CAC AAT GC-3´
Temperature profile:
94º - 2 min / (94º - 1 min. / 55º - 1 min. / 72º - 1 min.) x 35 /
72º - 5 min.
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