By Annette M. Griffin, Hugh G. Griffin
DNA Sequencing Protocols provide you with the information to turn into a sequencing specialist. An all-star forged of investigators covers almost 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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Sterile, detergent-free 1-L conical flasks fitted with porous tops or cotton-wool bungs and an orbital incubator capable of shaking these vigorously (around 250 rpm) at 37°C. 4. , disposable Falcon 2070 or reusable Oakridge, sterile and detergent-free), and a centrifuge, preferably refrigerated, capable of spinning these tubes at 4,000g. 5. Transformation buffers (see Note 2): a. 5-mL aliquots. When needed, thaw, dilute to 15 mL, filter, and chill on ice. b. TFB: 10 mM K-MES, 100 mM RbCl or KCl, 45 mMMnC12.
L-36 2. Bankrer, A. , Brown, C. , Chee, M. , Hutchison, C. , Martrgnetti, J. , Satchwell, S. , Weston, K. , and Barrell, B. G. (1991) The DNA sequence of the human cytomegalovirus genome. DNA Sequence 2, l-12. 3 Davrson, A. J (1991) Experience m shotgun sequencmg a 134 kilobase pair DNA molecule. DNA Sequence 1,389-394. 4. Eperon, I. C (1986) Rapid preparation of bacteriophage DNA for sequence analysis in sets of 96 clones, using filtration. Anal. Biochem. 56,406-412. , Brown, C. M , Bankrer, A.
5% NaCl. 2. , Cornmg cell wells) There appears to be little advantage to using either tissue culture treated or nontreated plates. 3. Rigid plastic “sandwich” boxes (with lids) approx dimensions 12 x 18 x 6 cm. These are used simply as a convement holder for microttter trays, providmg sufficient air supply for good phage growth but maintaming an isolated environment. The trays can be held m position within the boxes using blocks of foam sponge. 4. Sterile, uncoated, wooden toothpicks. 5. C. Centra 4X).