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Additional info for The QIAGEN Guide to Template Purification and DNA Sequencing
Sample text
D. d. d. d. d. d. d. d. d. d. d. d. d. d. d. d. d. d. d. Not determined * † ‡ © 1998 QIAGEN XII. qxd 3/6/98 17:06 Uhr Page 52 E. Sequencing reaction setup for resolving elongation stop structures* A* B* Setup 400 ng 1 µg 4–10 pmol 10 pmol Primer 4 µl 16 µl PRISM Ready Reaction dRhodamine Dye Terminator Premix 2 µl – 20 µl Template DNA Dimethylsulfoxide (DMSO) 40 µl Reaction volume F. Direct genomic DNA sequencing protocols The optimum amount of genomic DNA and the number of cycles are dependent on the genome size and may have to be determined for each bacterial genome.
Dissolve (stir and warm, not above 50°C) the acrylamide solution and deionize with Serva Serdolit for 10 min. 2 mm filter. Then pass the dissolved acrylamide solution through the same filter (combines acrylamide solution and 10x TBE), and degas for 1 min under vacuum. Add 350 µl ammonium persulfate (10%) and 15 µl TEMED for the polymerization (>3h). After 1 hour place a wet paper towel at the bottom of the gel to avoid the occurence of the “red rain” phenomenon. Add 350 µl ammonium persulfate (10%) and 15 µl TEMED for the polymerization (>3h).
B modified BigDye terminator sequencing protocol that can often resolve elongation stop structures. C slippage and stuttering effects of polymerase elongation due to a long poly-T stretch. Note that elongation stops can be more easily detected in the “full view” mode of a sequencing file (right panels of A and B ). VIII. qxd 18/6/98 9:29 Uhr Page 42 reducing sequence quality (Ref. 82, Figure 33, for modified protocol see Appendix). GC-rich templates should also be sequenced using dye-terminator chemistry, since prematurely terminated molecules, which constitute most of the spurious signal in a strong stop in a dye primer reaction, will not be detected when dye terminators are used (Figures 34A and 34B).