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    Sequencing Double-Stranded DNA Using the Sequenase Version 2.0 Kit

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    969

    实验步骤

     

    1. Prepare the DNA for sequencing as per the protocols, "Purification and Concentration of Aqueous DNA Solutions" and "Alkali Denaturation of Supercoiled Plasmid DNA".

    2. Warm a heating block up to 65 degrees.

    3. For each sample to be sequenced, put the following in a 1.5 ml Eppendorf tube:

    DNA (prepared as discussed) 7 ul

    Sequencing primer 1 ul

    Reaction Buffer 2 ul

    4. Heat to 65 degrees for 2 minutes, then remove heating block from heat source and allow to slowly cool to <35 degrees. Heating the DNA separates the strands and allows the primer to anneal as the temperature slowly declines.

    5. While cooling, prepare the following mixtures. Prepare slightly more than you actually need, in order to avoid problems during the actual procedure.

    6. Also at this time, prepare the microtiter dish that will be used during the termination reactions. Transparent tape should be applied over any wells that will not be needed, in order to provide a better seal against water leaking into the sample wells. The horizontal rows should be labelled A-T-G and C, while the vertical columns should be labelled with a code for your samples. When this is complete, place 2.5 ul of the appropriate ddNTP Termination Mix into each respective well. Use the appropriate set of Termination Mix tubes for either dGTP reactions (red-capped tubes) or for dITP reactions (green-capped tubes).

    Be sure the drop of Termination Mix makes it to the bottom of the well by tapping the plate upon the bench. Keep the plate loosely covered with a plate sealer until needed.

    7. Once the annealed template-primer has reached 35 degrees or lower, add 5.5 ul of the Master Mix by pipetting it onto the side of the sample tube and tapping it down to mix thoroughly. Before adding the Master Mix, it is a good idea to briefly spin down the samples to recover any solution clinging to the top or sides of the tube.

    Try not to contaminate the pipet tip with any sample, as it can be used to add Master Mix to all sample tubes, minimizing the amount of solid radioactive waste generated.

    8. Incubate at room temperature for 1 minute to allow for primer extension and labelling.

    9. Pipet the labelling reaction up and down to mix, and add 3.5 ul to each of the four wells on the microtiter plate containing the four termination mixes. One pipet tip can be used to add the reaction mix to the four wells by pipeting the sample onto the side of the well. The plate is then tapped on the bench causing the labelling reaction to mix with the termination mix. This procedure, once again, minimizes the amount of solid radioactive waste generated.

    10. Once all the reactions have been added to the termination mixes in the plate, cover the plate tightly with a plate sealer and place in a water bath at 37 degrees for 5 minutes.

    The water in the bath should be high enough to bubble around the wells when you look under the plate. During this step the chains are extended a little more and then terminated by the addition of a dideoxynucleotide.

    11. Wipe off the water from the outside of the plate with a Kimwipe and carefully remove the plate sealer, making sure that no drops of water fall into the wells.

    12. Add 4 ul of Stop Solution to each well by depositing the drop on the side of the well and tapping to mix.

    13. Samples may be stored, covered, at -20 degrees for up to 1 week before running on a gel.

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