DNA Sequencing Protocols by Annette M. Griffin, Hugh G. Griffin

By Annette M. Griffin, Hugh G. Griffin

DNA Sequencing Protocols will give you the information to develop into a sequencing specialist. An all-star solid of investigators covers nearly all facets, together with advancements in cycle sequencing, sequencing PCR items, sequencing lambda and cosmids, multiplex sequencing, direct blotting electrophoresis, sequencing of chemiluminescence, and automatic sequencing.

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Of 80 pL. Digest for 2 h at 37°C (see 2. Add 50 pL phenol/chloroform and vortex for 1 min before centrrfuganon at 10,OOOgfor 4 mm. Repeat the extraction with 50 pL chloroform. 3. Spin-dtalyze the sample on acolumn prepared from 500 pL of Sepharose slurry (see Chapter 19 and Note 5). L for digestion with Exo III, storing the remainder of the sample at -20°C. 2. Protecting the Primer Site with Thiophosphate 1. 1. , the restriction site closest to the primer site of the vector). 2. Add 10 pL 10X TM and 2 pL of thlophosphate-dNTPs, followed by 15 U Klenow fragment of DNA polymerase Incubate 30 mm at room temperature.

This IS sufficient to transfer Infected cells from the plaque to the new culture. 5 h (see Notes 3 and 4). 4. 5~mL microfuge tubes and spin for 5 min in a microfuge. 5. Transfer supernatants to clean tubes making sure the pellet is undisturbed, add 200 uL of PEG/NaCl to each, vortex well, and leave at room temperature for at least 15 min. 6. Spin for 5 min in microfuge to pellet the phage particles. Remove the PEG-containing supernatant with a pipet, respin the tubes for a few seconds and carefully remove all remaming traces of supernatant from around the phage pellet using a drawn-out Pasteur pipet (see Note 5).

23: DNA Sequencrng Protocols E&ted by. H. and A. , Totowa, 37 NJ 38 Tomley 2. Materials 1. An E. coli strain suitable for propagating bacteriophage Ml3 vectors Recommended strains mclude JMlOl, JM103, JM107, JM109, TGI, and TG2. 2. 2X TY broth: 1% tryptone, 1% yeast, 100 mM NaCl. Sterthze by autoclaving in 50-mL aliquots. 3. , disposable lo-mL Falcon tubes or glass/plastic universal bottles) and an orbital incubator for shaking the tubes vigorously at 37OC. 4. A mrcrofuge and 1S-mL mrcrofuge tubes.

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