By Kumaran Narayanan (eds.)
Bacterial man made Chromosomes, moment Edition expands upon the former variation with present, specified equipment constructed for operating with BACs. up to date chapters incorporated during this version current primary recommendations used for BAC development and characterization, complex techniques for introducing alterations, reaching gene expression from BAC vectors, purposes of BACs in version organisms, and clinical genetics and drug discovery. Written within the hugely profitable Methods in Molecular Biology sequence structure, chapters contain introductions to their respective issues, lists of the required fabrics and reagents, step by step reproducible laboratory protocols, and find out how to troubleshoot and stay away from identified pitfalls.
Authoritative and state of the art, Bacterial synthetic Chromosomes, moment variation seeks to help scientists in advancing their study utilizing those fascinating BAC thoughts and strategies.
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Additional resources for Bacterial Artificial Chromosomes
5 ml Eppendorf tubes. Add 100 μl of zymolyase solution, vortex the cells for 4 s, and incubate the suspension for 30 min at 37 °C. 4. Melt an appropriate quantity of LMP agarose and place it in a 50 °C water bath to cool. 5. Transfer the melted agarose and resuspended cells to a 42 °C heating block and equilibrate for 15 min. 6. Add to the cell suspension an equal volume of the melted agarose and mix well by vortexing. Keep the cell/agarose suspension at 42 °C. 5 %. With a higher concentration of agarose it is impossible to completely melt the plugs for electroporation.
26. 27. 28. 29. 30. 31. 32. 25 mere for correction of genetic deficiencies in human cells. Proc Natl Acad Sci U S A 108:20048–20053 Kouprina N, Ebersole T, Koriabine M, Pak E, Rogozin IB, Katoh M, Oshimura M, Ogi K, Peredelchuk M, Solomon G, Brown W, Barrett JC, Larionov V (2003) Cloning of human centromeres by transformation-associated recombination in yeast and generation of functional human artificial chromosomes. Nucleic Acids Res 31:922–934 Kouprina N, Pavlicek A, Noskov VN, Solomon S, Otstot J, Isaacs W, Carpten JD, Trent JM, Barrett JC, Jurka J, Larionov V (2005) Dynamic structure of the SPANX gene cluster mapped to the prostate cancer susceptibility locus HPCX at Xq27.
Incubate the culture at −20 °C for 20 min, swirling the bottle gently at 3-min intervals. 20. Centrifuge the bottle at 6,800 × g, −2 °C, for 12 min. 21. Sit the bottle on ice, and carefully remove the broth, leaving the pellet intact at the bottom of the bottle. 22. Gently but thoroughly resuspend the cell pellet in 250 mL of ice-cold 10 % (v/v) glycerol using a precooled 10 mL serological pipette. 23. Centrifuge the resuspended culture at 6,800 × g, at −2 °C, for 12 min. 24. Repeat the glycerol washing steps with 250 mL of ice-cold 10 % (v/v) glycerol (see steps 21–23) twice.