Plant Virology Protocols: From Virus Isolation to Transgenic by Gary D. Foster, Sally Taylor

By Gary D. Foster, Sally Taylor

A finished selection of state of the art options for producing transgenic crops which are immune to plant viruses through the cloning and expression of the coat protein gene. Its unfailingly reproducible tools, perfected by means of hands-on masters, conceal the full procedure from virus isolation, RNA extraction, and cloning coat protein genes, to the creation of the coat protein gene into the plant genome and the trying out of transgenic crops for resistance. equipment for checking out for transformation through PCR and Southern blotting, the detection of RNA transcripts via Northern blotting, and the construction of protein via Western research are supplied, as are tools for difficult the transgenic crops produced and for detecting and measuring the degrees of virus.

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Additional info for Plant Virology Protocols: From Virus Isolation to Transgenic Resistance (Methods in Molecular Biology)

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B 8pL125mMdNTPs c. 1 pL 5’ Sense-strand primer (100 pmol/l pL). d 1 pL 3’ Antisense-strand primer (100 pmol/l pL) e. 30 pL Sterile H20. f 1 pL Tth DNA polymerase (4U/uL) g. 5 pL First-strand cDNA made from up to 1 ug dsRNA. Overlay the reaction mixture with one drop (about 30 &) of light mineral oil and briefly spm Carry out the amplification of cDNAs with a thermal cycler Typical conditions for the synthesis of cDNA, denaturation, annealmg, and polymerization are as follows 9 10 11 12 13 14 15 16 17 18.

Covey et al. 56 3. Sterilized glass rod with tip flattened obliquely 4. Sodium phosphate buffer, pH 7 2 Stock solutions, 0 5M Na2HP04 (17 91 g/100 mL), 0 SMNaH,PO, (7 8 g/100 mL) For 200 mL of buffer, mix 144 mL of 0 5M Na,HP04 with 56 mL 0 5M NaH,PO,, cool to 4’C 5 Solid sodium sulfite (0 75%). Weigh out 1 5 g per 200 mL extraction buffer 6 Solid urea (1M). Weigh out 12 g for 200 mL buffer 7. 10% Trlton X-100 (stock solution) 8. DNase buffer. 50 mMTris-HCl, pH 7 5, 5 mA4MgC1, 9. Phenol:chloroform mixture.

DNase buffer. 50 mMTris-HCl, pH 7 5, 5 mA4MgC1, 9. Phenol:chloroform mixture. 1). Caution: Phenol*chloroform 1s extremely corrosive and toxic. It IS best bought as a preprepared solution; handle m small volumes with great care 10 10 mg/mL Protemase K (Boehrmger) m TE with 1% SDS 11. 2 mg/mL Deoxyrlbonuclease I (DNase I, Sigma) m DNase buffer 12 2 mg/mL Pancreatic rlbonuclease A (RNase A) m TE (heat-treated by mcubatlon at 95°C for 10 mm) 13 10% Sodium dodecyl sulfate (SDS) 14 TE solution 15. 0 5M MgCl, 16 30% Polyethylene glycol 6000 (PEG) 17 Centrifuge rotors cooled to 4°C.

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