DNA sequencing is the determination of the precise sequence of nucleotides in a sample of DNA. The most popular method for doing this is called the dideoxy method.
The equipment and supplies:
·Sterile 1.5 mL and 0.2 mL microcentrifuge tubes and racks
·Ice
· Micropipettes, 0.5 to 1.0 µl, 10 to 100 µl, 100 to 1000 µl
·Sterile pipette tips
·Disposable gloves, safety glasses, and lab coats
·Microcentrifuge, for 1.5 ml and 0.2 ml PCR (adapter) microcentrifuge tubes
·Sterile deionized water
·Template DNA
·Primers (Always determine the concentration of primer stock by reading at 260 nm (OD260).
·PCR machine (Thermocycler)
·7.5 Molar Ammonium acetate aqueous solution
·Freezer, -20°C
·Water bath set at 70°C
·37°C incubator
·Sterile gel loading pipet tips
·UV-box for photo-polymerization
·Lint-free medical wipes (Kimwipes or similar)
·Thermo Sequenase Cy5 Dye Terminator Cycle Sequencing Kit Amersham Biosciences (#27-2682-01), store at -20ºC
·Alfexpress II DNA Sequencer from Amersham Biosciences
·Long Read Gel Kit
·TBE Buffer
Protocol
**Important notes before you begin: All reagents should be kept on ice when removed from storage for use. Also please be aware that repeated freeze-thawing of the Cy-labeled terminators is not recommended. All reagent tubes should be centrifuged before opening them. Whenever possible, keep the tubes capped and on ice to minimize evaporation of the small volumes of the reagents used. Thoroughly mix all reaction mixtures after each addition and centrifuge tubes briefly to collect the reaction mixtures at the bottom of the tubes. Dispense all reagents carefully with new tips for each transfer to avoid contamination of stock solutions.
***Safety Warnings and Precautions: All chemicals should be considered as potentially hazardous. Wear suitable protective clothing such as lab coat, safety glasses, and gloves. Care should be taken to avoid contact with skin or eyes. This protocol requires the use of ethanol and formamide. Gel reagents may contain acrylamide, a neurotoxin and suspected carcinogen. Please follow the manufacturer’s Material Safety Data Sheet regarding safe handling and use of these materials.
A. Preparation of ddA, ddC, ddG, ddT termination mixes (Estimated Time 15-30 min.)**Only one set of termination mixes are required to sequence up to 10 DNA templates—DO NOT set up termination mixes for each template!
1.Label four 1.5 mL microfuge tubes “ddA, ddC, ddG, ddT” for the respective termination mixes.
2.To each tube, add the components indicated below to prepare the dNTP/Cy5 ddNTP termination mixes, beginning with the water first. The volumes are sufficient for 10 DNA template sequencing reactions.
3.Flick the tubes gently to mix—do not vortex. Centrifuge the tubes briefly (10 sec. at max rpm) to collect the contents at the bottom of each tube.
4. Store the dNTP/ Cy5-ddNTP mixes on ice and in the dark until needed. (Cover tubes with aluminum foil).
B. Preparation of Master Mix (Estimated Time 15-30 minutes)
NOTE: Make sure you have a water bath heating to 70ºC while mixes are being prepared. It will need to be up to temperature prior to loading samples for sequencing.
5. Label one 1.5 mL microcentrifuge tube with the DNA sample name for each DNA template with primer to be sequenced. Keep tubes on ice and capped when not in use. Ex. “DNA#1 Forward Master Mix”, “DNA #1 Reverse Master Mix”, “DNA #2 Forward Master Mix”, etc.
6. Using the table below, each group will prepare master mixes for each DNA template with primer to be sequenced. Begin by adding the water first, Reaction Buffer, DNA and primer, and Thermo Sequenase polymerase last. **Important: The Thermo Sequenase polymerase must be kept on ice and capped whenever possible. Handling time must be kept brief.
