Cotton ovules (Gossypium hirsutum cv. Coker 312) were collected 8, 15, and 20 days after anthesis. Total RNA was extracted from stripped ovules (fibers removed) and isolated fibers using the procedure of John (1992). - RNase-free DNase I (1000 units/ml, Promega, Madison, WI)
- RNasin (recombinant ribonuclease inibitor, 2600 units/ml, Promega, Madison, WI)
- DNase I buffer: 100 mM Tris-HCl (pH 8.0), 25 mM NaCl, 15 mM MgCl2, 0.25 mM CaCl2, 2.5 mM KCl
- PCI: phenol:chloroform:isoamyl alcohol (25:24:1)
- CI: chloroform:isoamyl alcohol (24:1)
- 7.5 M ammonium acetate
- Moloney murine leukemia virus reverse transrciptase M-MuLV RT (20000 units/ml, Promega, Madison, WI)
- 5X M-MuLV RT buffer (Promega, Madison, WI)
- 20 uM decamer primers (random)
- 20 uM anchored oligo-dT12MN primers (M=A,C, or G; N=A,C,G, or T)
- mixed dNTPs (2.5 mM each dNTP, 100 uM each dNTP
- Taq DNA Polymerase and buffer (Promega, Madison, WI)
- 25 mM MgCl2
- alpha-[33P]-dATP (NEN Research Products, du Pont de Nemours & Co, Boston, MA)
- 40% acrylamide:bisacrylamide (29:1) stock solution (Fisher Scientific, Houston, TX)
- TBE buffer: 0.09 M Tris-borate, 0.002 M EDTA (pH 8.0)
- sample loading solution: 0.25% bromophenol blue, 0.25% xylene cyanol FF, 40% (w/v) sucrose in water
- TA cloning kit (pCRII vector system, Invitrogen, San Diego, CA, or pGEM-T vector system, Promega, Madison, WI)
- TAE buffer: 0.04 M Tris-acetate, 0.001 M EDTA (pH 8.0)
- To a 1.5 ml microcentrifuge tube, add 30 to 50 ug of RNA, 40 ul of DNase I reaction buffer, 5 ul RNasin, 10 ul DNase I, bring to total volume of 100 ul with DEPC treated water, mix gently.
- Incubate 30 min in a 37C water bath.
- Purify the RNA by extraction with PCI and CI.
- Add 1/2 volume of 7.5 M ammonium acetate, mix well.
- Add 2 volume of cold ethanol, precipitate the RNA at -20C for at least 1 hour.
- Centrifuge at 10000X g for 15 min at 4C, discard ethanol, air dry the pellet, and resuspend in 40 ul DEPC-treated water.
- To a 0.5 ml microcentrifuge tube, add 2 ug of DNase I-treated total RNA, bring to 25 ul with DEPC treated water.
- Heat the tube in 65C water bath for 10 min.
- Add 10 ul 5X M-MuLV RT buffer, 5 ul 2.5 mM mixed dNTPs, 6 ul 20 uM anchored oligo-dt primer, 1 ul RNasin.
- Incubate at room temperature for 15 min.
- Add 1.5 ul M-MuLV RT, gently mix.
- Incubate at 37C for 1 hour.
- Heat the reaction in 95C water bath for 5 min, store cDNA at -20C for later use.
- To a 0.5 ml microcentrifuge tube, add 4.0 ul cDNA, 2.5 ul 100 uM mixed dNTPs, 2.5 ul 10X Taq DNA polmerase buffer, 2.5 ul 25 mM MgCl2, 2.5 ul 20 uM anchored oligo-dT primer (same as was used in the cDNA synthesis), 1.0 ul 20 uM 10-mer arbitrary primer, 0.5 ul alpha-[33P]dATP (10 mCi/ml), 0.5 ul Taq DNA polymerase, bring the reaction to 25 ul with sterile water, gently mix, cover the reaction with 25 ul sterile mineral oil (if needed).
- Run PCR reaction as follows: 94C for 30 sec, 42C for 1 min, 72C for 30 sec, 40 cycles, followed by a 5 min final extension at 72C.
- Pour a 6% non-denaturing polyacrylamide sequencing gel (same as regular sequencing gel but without urea).
- Pre-run the gel in 1X TBE buffer at 30 mA for 30 min.
- Add 8 ul sample loading solution to each reaction, mix well, heat at 72C water bath for 2-3 minutes prior to loading.
- Run the gel at 30 mA until xylene cyanol reaches the bottom of the gel.
- Dry the gel without fixing, tape the dried gel and X-ray film together, mark the film and the gel by punching several holes with a needle.
- Expose the film for 8-10 hours (or overnight) at -80C with intensifying screens.
- Align the developed film with the gel, cut differentially displayed bands from the dried gel with a razor blade, cut into small pieces, and put them into a 1.5 ml microcentrifuge tube.
- Add 200 ul sterile water (good for two 1.5mm x 7mm slices), incubate for 15 min at room temperature.
- Boil the tube for 10 min.
- Centrifuge 1 min at 10000X g, transfer the supernatant to a fresh tube, and pipette 10 ul into another fresh microcentrifuge tube.
- Reamplify this 10 ul of eluted DNA in a 25 ul reaction volume using the same primers and PCR conditions as used above, but without radiolabelled dATP.
- Remove 10 ul of reamplified PCR product, run on a 1% agarose gel in 1X TAE buffer, stain the gel with ethidium bromide (10 mg/ml).
- Ligate the remaining PCR products into a TA cloning vector (pCRII or pGEM-T), and transform competent Escherichia coli (e.g. DH5) cells.
