DNA Sequencing, 1st Edition by T.S. Work and R.H. Burdon (Eds.)

By T.S. Work and R.H. Burdon (Eds.)

This quantity presents a accomplished description of the rules and techniques utilized in DNA sequencing. Following a close creation the chapters are: DNA sequencing; Chain terminator sequencing; Primed synthesis tools utilized to DNA fragments cloned into phage M13; DNA sequencing by means of the Maxam-Gilbert chemical technique; machine equipment for DNA sequencers; Appendices together with contractions and specified phrases, cloning vectors, commercially to be had restrict endonucleases, and autoradiography.

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Load the samples into the gel wells using a drawn-out capillary tube. A suitable order is: - Run the gel at about 6OOV until the fast migrating dye (bromophenol blue) is at the bottom of the gel (approx. 4 hr). The gel gets quite hot during electrophoresis ensuring the DNA remains fully denatured. - Remove one glass plate from the gel and cover the exposed surface with cellophane film. ). Alternatively the gel may be fixed by immersion in 10% acetic acid for 15-20min, washed 1-2 min in distilled water, blotted dry with absorbent paper, covered with cellophane and autoradiographed at room temperature.

Will yield molecules with clean blunt-ended termini which can be Ch. 1 INTRODUmON 29 inserted into the vector either directly, using blunt-ended ligation, or after the addition of artificial linkers to produce cohesive ends (Chapter 4). 14. Conclusion All these methods aim at producing a recombinant DNA which can be propagated and amplified at will in order to prepare a sufficient quantity of DNA for sequencing. Amplification in plasmids and phage A produces double-stranded recombinant DNA from which the segment to be sequenced can usually be excised using restriction endonucleases.

Keep at 0°C. 5. 2(ii). 2(ii). Step 5(i): Cleavage at ribosubstituted site. (i) Cleavage with pancreatic ribonuclease - The eight plus and minus samples and an aliquot (6p1) of the original extension product from Step 3 are sealed in their capillary tubes. - Heat 100°C for 3 min to denature the DNA. - Cool rapidly in ice-water. - Open the tubes and add, with mixing, 1 p l of a 10mg/ml solution of ribonuclease A. - Incubate at 37°C for 30 min. - Stop the reaction by adding 10 p1 formamide-dye mix at 0°C.

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