- Linearize 25&g of template DNA in a volume of 50&l with 20U of the appropriate enzyme in a 0.5ml Epp. Perform the reaction O/N at 37°c. Complete linearization of the DNA is essential as circular DNA is a much better template for transcription than linear DNA.
- Carry out an extraction with 30&l of phenol/chloroform. Take 45&l of the aqueous phase and spin dialyse through Sepharose CL-6B in a 0.5ml Epp. Check that the DNA has been efficiently linearized by electrophoresis of a 1&l aliquot on a 1% agarose/TBE gel.
- Set up the transcription as indicated below. The order in which the reactants is added is important because the RNase inhibitor will release RNase in the absence of DTT and secondly because the spermidine in the T7 buffer can precipitate DNA. To prevent DNA precipitation the reaction components should be mixed together at room temperature.
10&l 5X T7 buffer (2)
2.5&l 0.1M DTT
1&l RNase inhibitor (3)
5&l 10X A/C/U/G mix (4)
5&l 5mM cap (5)
10&l linear DNA
- Incubate reaction for 5' at 37°c, add 5&l of T7 (6) and mix by pipetting up and down.
- Incubate reaction for 25' at 37°c and then add 5&l of 20mM GTP. Do not mix vigorously.
- Incubate reaction for 35' at 37°c. Carry out a phenol/chloroform extraction with 30&l. Take 45 &l of the aqueous phase. Set aside 2&l of the transcripts for running on a gel. Either use the remainder of the transcripts immediately or ethanol precipitate them.
- Transcripts can be run on an ordinary 1X TBE gel alongside virion RNA standards. For "midi gels" a voltage of 80V is suitable. Running the bromophenol blue dye half-way down the gel will ensure separation of the DNA and
- All solutions and equipment should be RNase free.
Therefore solutions should be made up with DEPC treated water; all tubes and tips should be sterile and untouched by human hands; gel running equipment should be treated with 0.5M NaOH to destroy RNase.
- Supplied by BRL: 5X buffer is 200mM Tris-HCl pH 8.0, 40mM MgCl2, 10mM spermidine, 125mM NaCl.
- 37-40U/&l from Boehringer or Pharmacia.
- 10X A/C/U/G mix is 20mM of each ATP, CTP, UTP and 2mM GTP.
- m7G[5']ppp[5']G from Boehringer or Pharmacia. Dissolve 5U in 60&l.
- 50U/&l from BRL.
Modifications to Promega Transcription Protocol for Protoplasts
After 100ul transcription reactions as above
- Extract once with equal volume phenol:chloroform.
- Recover s/n and add equal volume of 4M LiCl.
- Leave on wet ice for 30' (this is more than enough - transcript is 95% recoverable after only 15'),
- Pellet transcripts in microfuge (5').
- Discard s/n and r/s pellet - which is generally NOT readily seen - in 100ul DEPC water.
- Spin dialyse resuspended transcripts to free of residual LiCl.
PVX inoculations on tobacco
All quantities given are for inoculation of one plant.
- Dissolve the RNA in DEPC treated water 20 microL maximun. If the inoculum is DNA use about 10 microgrammes in this volume.
- Add 1M sodium phosphate pH 7.0 to a final concentration of 42.9mM , vortex and flick spin.
- Add bentonite (10-20%), pipette up and down a few times to mix (Optional).
- Take tobacco plant ( 4-6 leaf plant ).
- Mark two adjacent leaves for inoculation with a Gilsen tip. Inoculating two adjacent leaves means that the next leaf above and between them will be infected systemically.
- Lightly dust the leaves to be infected with carborundum from a plastic water bottle ( silicon carbide, 600 grit, BDH ).
- Using a Gilsen pipette 10-20&l of solution, in small drops, onto each leaf to be infected.
- Wearing gloves rub the leaves of the plants, covering all of each of the leaves.
- Leave plants for 5' then water off the carborundum.
- Recalcitrant plants can be softened up by leaving them in the dark for 24 hours prior to inoculation.
Detection of GFP
- in whole plants. The plants were illuminated with a 100W hand-held U.V. lamp (U.V. Products of Upland Ca 91786, Blak Ray model B 100AP) and photographed with Kodak Ektachrome Panther (400 ASA) film through a Wratten 58 (green) or 8 (yellow) filter. Exposure times varied up to 70sec depending on the intensity of the fluorescence and the distance of the camera and lamp from the plant .
- by epifluorescence microscopy. Whole leaves were viewed using an epifluorescence microscope (Optiphot, Nikon) using the following Nikon filters: U.V. (excitation filter 330-380 nm, barrier filter 420 nm, dichroic mirror 400 nm); BV (excitation filter 400-440 nm, barrier filter 470 nm, dichroic mirror 455 nm). Photographs were taken using Kodak Ektachrome Panther (400 ASA) film.
- by confocal microscopy. A BioRad MRC1000 CLSM was used to image GFP fluorescence in whole leaves. A 25 mW Krypton/Argon laser (detuned to 15 mW) was used to produce blue excitation light at 488 nm (emission filter 522 nm). No significant autofluorescence was seen using these filter combinations. During observation individual images were stored on optical disc. Stored images were printed using a Kodak XLS digital printer.
- by fluorescence spectroscopy. Plant tissue was ground with a pestle and mortar in two volumes of extraction buffer (50mM Tris-HCl pH 8.0, 10mM EDTA, 10mM dithiothreitol, 18% glycerol) and filtered through Miracloth (Calbiochem). The filtrate was centrifuged to pellet debris and the supernatant collected. The supernatant was diluted ten fold with buffer and purified through a PD10 column (Pharmacia), which had been equilibrated in buffer. The protein content of the extracts was determined by the method of Bradford (Bradford, 1976) using a kit from BioRad Laboratories. Preparations were diluted with buffer to 80&g per ml and analyzed in a Kontron SFM25 scanning fluorometer.
- by electrophoresis. Plant extracts were prepared as described above, but without column purification and dilution. Samples were electrophoresed, without prior heat treatment, on 12.5% polyacrylamide SDS-PAGE gels according to Laemmli (Laemmli, 1970). GFP was detected by illumination with a 100W hand-held U.V. lamp as described above for the infected plants.