By Kumaran Narayanan (eds.)
Bacterial synthetic Chromosomes, moment Edition expands upon the former variation with present, distinct tools constructed for operating with BACs. up to date chapters integrated during this version current basic concepts used for BAC building and characterization, complicated methods for introducing ameliorations, 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 layout, chapters comprise introductions to their respective themes, lists of the required fabrics and reagents, step by step reproducible laboratory protocols, and the way to troubleshoot and keep away from recognized pitfalls.
Authoritative and state of the art, Bacterial synthetic Chromosomes, moment variation seeks to help scientists in advancing their examine utilizing those intriguing BAC innovations and strategies.
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Additional resources for Bacterial Artificial Chromosomes
5) supplemented with 5 mM of DAP and zeocin at 30 °C with 150 rpm shaking for 30–48 h. 2. Use 1 μL aliquot of the asd− zeor tos+ cmr DH10B culture as template to obtain DNA. Prepare the following mixture in a PCR tube for each reaction (see Note 10). Recombineering Linear BACs No. 5 Total 50 33 Volume (μL) 3. Subject the mixture to the following cycling condition: Step Temperature (°C) Duration (s) 1 2 3 4 5 6 95 95 45 68 68 4 120 30 30 120 600 Forever 35 cycles 4. Electrophorese the PCR product in a 1 % (w/v) agarose gel (see Note 11).
2. Once verified, precipitate the DNA. 2) and 250 μL of −20 °C absolute ethanol. Recombineering Linear BACs 43 3. Shake the mixture vigorously and incubate at −80 °C for 30 min (see Note 36). 4. Centrifuge at 15,000 × g for 15 min at room temperature. 5. Wash the pellet with 250 μL of 70 % ethanol and centrifuge at 15,000 × g for 5 min at room temperature. 6. Remove the remaining supernatant with a micropipette tip and let the pellet air-dry for 5–10 min. 7. Resuspend the pellet in 5–10 μL of TE buffer.
D) In vivo linearization of BAC-tos. The circular BAC-tos is transformed into a telN+ E. coli constitutively expressing the TelN protein, resolving the tos sequence into individual left (telL) and right (telR) telomeres and generating a linear BAC-tos . (e) Extraction of linear BAC-tos. Linearized BAC-tos DNA is then column extracted for downstream applications. (f) Detecting presence of telomeres. To test for the integrity of telomeric ends formed on the linearized BAC-tos, the DNA is incubated with RecBCD exonuclease V that degrades open-ended linear DNA.
Bacterial Artificial Chromosomes by Kumaran Narayanan (eds.)