Biotech Glossary | Bioinformatics | Lab Protocol | Notes | Malaysia University |
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Double Stranded DNA Sequencing Template Preparation |
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| Procedure |
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1. In an empty tube, combine 6 μg of template DNA with 6 μl 1 M NaOH and increase the volume to 24 μl with ddH2O. 2. Denature the DNA sample at 85°C for 5 min, then place the tube in an ice bath. Add 3 μl of ice-cold 2 M Ammonium Acetate and 3 μl 70% Ethanol. Incubate the tube at -20°C for 1 to 2 hr. 3. Centrifuge for 10 min at high speed in a microcentrifuge. Rinse the DNA pellet with 70% Ethanol and centrifuge again for 10 min at high speed in a microcentrifuge. 4. Pour off the Ethanol and dry the DNA pellet in a speedvac concentrator for 30 min (see Hint #1). |
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| Solutions |
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| BioReagents and Chemicals |
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Sodium Hydroxide Ethanol Ammonium Acetate |
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| Protocol Hints |
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1. Drying is important, as any residual Ammonium Acetate will affect the gel. |
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