By Michael M. Weil, Rashmi Pershad, Ruoping Wang (auth.), Shaying Zhao, Marvin Stodolsky (eds.)
The skill to move and retain DNA either inside and among species is a vital ability in biotechnology and drugs. In Bacterial man made Chromosomes, specialist investigators describe not just the vintage equipment, but in addition the numerous novel thoughts they've got perfected for the move of enormous DNAs into the cells of either microbes and animals through large-insert recombinant DNAs. quantity 2: practical stories presents a large choice of equipment and functions for useful research of the DNA-transformed organisms. The laboratory protocols stick to the profitable tools in Molecular Biology™ sequence layout, each providing step by step laboratory directions, an advent outlining the primary in the back of the approach, lists of kit and reagents, and tips about troubleshooting and heading off recognized pitfalls. along with protocols, every one bankruptcy contains medical reports, software program instruments, database assets, genome sequencing concepts, and illustrative case stories. An accompanying quantity, quantity 1: Library building, actual Mapping, and Sequencing, offers simply reproducible ideas for library building, actual mapping, and sequencing.
complete and state-of-the-art, the 2 volumes of Bacterial man made Chromosomes offer a superlative selection of hugely efficient protocols that may end up helpful to many bioscientists, together with genome sequencers, geneticists, molecular biologists, and biochemists learning the constitution and serve as of particular genomes.
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Additional info for Bacterial Artificial Chromosomes: Volume 2 Functional Studies
6. 5 µL of 10 mM ATP solution into each tube of an 8-tube strip (see Note 16). Cap the 8-tube strip and place on ice. 7. , step 5). 5 µL of 10 mM ATP and dispense 1 µL on the inside wall of each of the 96 tubes containing DNA. Gently tap the PCR rack so that the ATP slides into the DNA/primer solution. Seal with a 96-well plate seal. This procedure for adding the ATP reduces the probability of carry over contamination. 8. Dispense 1 µL (5 U) of the T4 DNA ligase enzyme into each tube of the 96 tubes individually (see Note 12).
Move on to the next 8-tube strip. 4. Place the 8-tube strips, now containing 99 µL PCR mixture and 1 µL ligation mediated PCR product, in a PCR machine: 95°C for 10 min; 45 cycles at 95°C for 50 Snijders et al. 30 s, 50°C for 30 s, 72°C for 2 min, and a final extension at 72°C for 7 min, followed by an incubation at 4°C. 5. 5 µg/mL ethidium bromide along with a ϕ X174 RF DNA/Hae III Marker to check fragment length. Size of the PCR product should range from 200 to 1500 bp (see Note 19 and Fig. 3).
5. , autoclaved, deionized, and filtered water). 6. Ultrapure agarose (Invitrogen). 7. 008 M ethylenediamine tetraacetic acid (EDTA). 8. 1% ethidium bromide solution. Ethidium bromide is a mutagen. Always wear protective clothes when handling any form of ethidium bromide (see Note 4). 9. 25% xylene cyanol, 30% glycerol in H2O. Store at 4°C. 10. ϕ X174 RF DNA/Hae III Marker (Promega). 11. UV transilluminator. 12. 96-well plate seal (USA Scientific). 13. 5 µL). 2. Ligation of Specific Primers to BAC DNA 1.