By Gary D. Foster, Sally Taylor
A finished number of state of the art innovations for producing transgenic crops which are immune to plant viruses through the cloning and expression of the coat protein gene. Its unfailingly reproducible equipment, perfected through hands-on masters, hide the whole procedure from virus isolation, RNA extraction, and cloning coat protein genes, to the advent of the coat protein gene into the plant genome and the trying out of transgenic vegetation for resistance. equipment for trying out for transformation by way of PCR and Southern blotting, the detection of RNA transcripts by means of Northern blotting, and the construction of protein by means of Western research are supplied, as are equipment for difficult the transgenic crops produced and for detecting and measuring the degrees of virus.
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Additional resources for Plant Virology Protocols: From Virus Isolation to Transgenic Resistance (Methods in Molecular Biology)
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. 19 20. 2. Single-Step RT-PCR 1 In a OS-mL mtcrocentrtfuge tube, mix m the followmg order a 5 $, 10X amplification buffer b.
Rice Dwarf Vu-us 1 Virus source Infected race leaves and leaf sheaths showmg clear symptoms l-2 mo after Inoculation 2 0 1M Phosphate buffer, pH 6 0, contammg KH2P04 and Na,HPO, 3. Trrton X- 100 4. 0 7. 2. Rice Black-Streaked Dwarf and Rice Ragged Stunt Wruses 1. 2 3. 4. Extraction (GMT) buffer. 3. 1. Reagents for General Use 1. Mtllt-Q grade autoclaved H20. 2. TE. 0 pH 7 5. Uyeda et al. 68 3. 1) 5 3M Sodmm acetate, pH 5 2. 2. sRNAs from Plants 1 2 10X STE buffer lMNaC1, 100 mMTrts-HCl, pH 6 8, 10 mMEDTA.
Trrton X- 100 4. 0 7. 2. Rice Black-Streaked Dwarf and Rice Ragged Stunt Wruses 1. 2 3. 4. Extraction (GMT) buffer. 3. 1. Reagents for General Use 1. Mtllt-Q grade autoclaved H20. 2. TE. 0 pH 7 5. Uyeda et al. 68 3. 1) 5 3M Sodmm acetate, pH 5 2. 2. sRNAs from Plants 1 2 10X STE buffer lMNaC1, 100 mMTrts-HCl, pH 6 8, 10 mMEDTA. 1X STE buffer Dilute the 10X STE buffer wtth stertle Hz0 3 CC41 cellulose powder (Whatman). 3. Drect Extract/on of Viral mRNAs and Genomlc dsRNAs from Plants 1 Mrlh-Q grade Hz0 treated wtth 0 1% DEPC and autoclaved 2 RNA extraction solutton: 4M guanidimum thiocyanate, 25 mM sodium citrate, pH 7 0,O 5% sarcosyl, 0 1M2-mercaptoethanol This solution is made by mixing the followmg a 250 g Guamdmmm thtocyanate (Fluke) IS dtssolved m 293 mL Mtlh-Q grade H20 b 17 6 mL 0 75M Sodmm cttrate, pH 7 0 c 26 4 mL 10% Sarcosyl The stock solution (a + b + c) can be stored for at least 3 mo at room temperature d 0 36 mL 2-Mercaptoethanol/SO mL RNA extraction solutton (a + b + c + d) can be stored for 1 mo at room temperature 3 Phenol (nucleic acid grade) Equrhbrate phenol with Milh-Q grade H20 4 2M Sodmm acetate, pH 4 0.