Plant Molecular Biology by Karam B. Singh (auth.), Stanton B. Gelvin, Robbert A.

By Karam B. Singh (auth.), Stanton B. Gelvin, Robbert A. Schilperoort, Desh Pal S. Verma (eds.)

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6. Heat at 75 °C for 30 min, vortex every 5-10 min. 7. m) and spin for 1-2 min in a microfuge. 8. 5 to the filtrate and precipitate with 3 volumes of ethanol in a dry ice-ethanol bath. 9. Centrifuge 30 min. Rinse with 80% ethanol and dry. 10. l of TE and quantitate by measuring the A260. 05% xylene cyanol. 15% polyacrylamide gel: 8 M urea, 15% polyacrylamide:bisacrylamide (38: 2). 1 x TBE, (200 x 400 x 1 mm, slot sizes: 20 x 5 x 1 mm). 3. 5 mM EDTA. TE: 10 mM Tris-HCI pH 8, 1 mM EDTA. PMAN-818/19 Hybridization and elongation of primer Steps in the procedure 1.

L of oligonucleotide elution buffer. 6. Heat at 75 °C for 30 min, vortex every 5-10 min. 7. m) and spin for 1-2 min in a microfuge. 8. 5 to the filtrate and precipitate with 3 volumes of ethanol in a dry ice-ethanol bath. 9. Centrifuge 30 min. Rinse with 80% ethanol and dry. 10. l of TE and quantitate by measuring the A260. 05% xylene cyanol. 15% polyacrylamide gel: 8 M urea, 15% polyacrylamide:bisacrylamide (38: 2). 1 x TBE, (200 x 400 x 1 mm, slot sizes: 20 x 5 x 1 mm). 3. 5 mM EDTA. TE: 10 mM Tris-HCI pH 8, 1 mM EDTA.

Better amplification results were obtained when oligonucleotides were purified prior to use. Steps in the procedure 1. l of loading buffer. Load the sample on a 15% polyacrylamide gel. 2. Run the gel at 45 Win 1 x TBE until the xylene cyanol has migrated ca. 12 em (this is suitable for 11 to 45 mers). 3. Remove the top glass plate and cover the gel with saran wrap. 4. Place above a silica gel plate (TLC F254, Merck) under UV-Iight and cut out with precision the oligonucleotide using a razor blade.

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