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| PREPARATION OF PLASMID DNA FOR SEQUENCING
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| [ 文章来源：omrf.ouhsc.edu | 文章作者：
| 发布时间：2007-01-12|
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The following protocol is based on our modifications of R. Kraft, J. Tardiff, K. S. Krauter, and L. A. Leinwand. Biotechn. 6(6):544-545, 1988.
- Inoculate 2-5 ml of L broth containing the appropriate antibiotic from a single bacterial colony. Incubate at 37°C overnight with vigorous shaking.
- Follow the Plasmid Quick Prep protocol through the potassium acetate step to isolate plasmid DNA.
- Add DNAse-free RNAse to 50µg/ml, incubate, 37°C from 10-30 minutes.
- Phenol extract the solution, saving the upper aqueous phase. Ether extract the remaining phenol, and ethanol precipitate the DNA. Resuspend the pellets in 16.8 µl H2
O, 3.2 µl 5 M NaCl, 20µl 13% PEG 8000, and incubate on ice for 30 min. Spin in a microfuge for 10 min, 4°C, and rinse the pellets once with 70% ethanol. Respin the pellets for 1 min and dry under vacuum.
Resuspend pellets in 20µl H2O and 2 µl (1/10 volume) of a solution of 2 N NaOH and 2 mM EDTA. The original protocol called for a 5 min room temperature incubation, others incubate 30 min @ 37°C, and Darise Farris reports good results at 85°C for 5 min then cooling on ice.
Neutralize with 1/10 volume of 3 M sodium acetate (pH 4.5-5.5) [or add 8 µl of 1 M Tris-HCl (pH 4.0-4.5), 3 µl of 3 M sodium acetate (pH 5.2)], and 75 µl of cold ethanol. Incubate on dry ice for 20 min.
Centrifuge at room temperature or 4°C in a microfuge for 5 min. Discard the supernatant and wash the pellet with 70% ethanol and centrifuge 2 min. Discard the supernatant and dry the pellet under vacuum.
Resuspend the pellet in 7 µl H2O, 1 µl of pUC (or the appropriate) primer at a 0.5 pmol/µl stock concentration, and 2 µl of 5x Sequenase buffer. Mix and centrifuge to remove any debri. Heat 2 min, 65°C and cool slowly to below 35°C by removing the heat block with samples in it from the heater to the lab bench. This cooling typically takes 30-45 min. Thaw the isotope during this time. Place tubes on ice when they reach 35°C.
Follow the Sequenase protocol from this point. Darise Farris recommends the following modifications for optimizing for short sequences: dilute the labelling mix 1:20 instead of 1:5, add 1 µl of Mn buffer (0.15 M sodium isocitrate & 0.1 M MnCl2) at the annealing step, and incubate the stop reactions for 15 min instead of 5 min.
RECIPES
- L Broth (LB; Luria-Bertani)
- 10 g tryptone
- 5 g yeast extract
- 5 g NaCl
- 1 L water
- Autoclave
- RNAse (10 mg/ml)
- Dissolve RNAse A in water. Boil for 5 minutes. Store at -20° C. Some protocols also add an equal volume of 40% glycerol and a half volume of 0.5 M NaCl.
- 5 M Sodium Chloride
- 73.06 g NaCl
- Add NaCl gradually to 200 ml H
2O. QS to 250 ml with water and autoclave.
- 3M Sodium Acetate, pH 5.5
- 18.46 g sodium acetate
- Adjust pH with glacial acetic acid (>8 ml), QS to 75 ml with water and autoclave.
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DNA sequencing (dye term