Bacterial artificial chromosomes

From OpenWetWare

Revision as of 17:15, 26 October 2005 by Reshma P. Shetty (Talk | contribs)
(diff) ←Older revision | Current revision (diff) | Newer revision→ (diff)
Jump to: navigation, search

Contents

Existing vectors

BAC vectors

  • original BAC vectors are 7.4 kb, others are 8-9kb.
  • can clone 80-300kb fragments

pBAC108L

  • no selection system for inserts

pBeloBAC11

  • recommended by Tom, have this in the lab
  • screen for inserts via α-complementation (blue-white screening on IPTG/Xgal plates)

pBACe3.6

  • transferred the pUC-link and SacBII sequences from a PAC vector to pBAC108L. See below.

PAC vectors

  • derived from bacteriophage P1 vector
  • 15-16kb
  • positive selection for inserts via sacB the levansucrase gene which converts sucrose to levan which is toxic to E. coli
  • a pUC-vector-derived fragment is inserted into the cloning site which inactivates sacB and allows large amounts of vector to be purified. Your insert replaces this pUC vector thereby returning the vector to single copy. Must be careful to ensure that no uncut vector is present in cloning because it will transform as well as the insert containing vectors. Thus, the sacB marker only prevents against vector religation productions not intact vector.

Protocol notes

Putting the BAC into E. coli

  1. Transformation
    • has low efficiencies
    • works on any size
  2. In vitro packaging
    • package the vector inside λ bacteriophage particles and infect cells
    • limits the size of the DNA because of phage head stability. The phage head is not stable if the vector is too small or too large. Usually ~40kb insert with an 8-9kb vector works.

BAC purification

  • most purification protocols appear to be geared for preparation of genomic libraries for sequencing. It is not clear if we need such elaborate protocols for simple clonings.
  • Typical yields
    1. 1μg from 5mL LB culture sing alkaline BAC DNA purification
    2. 0.8μg from 1.3mL TB culture using Qiagen R.E.A.L.
    3. 4μg from 20mL LB culture using Qiagen-tip 20
    4. 100μg from 500mL LB culture using Qiagen-tip 500

References

  1. F. W. Whipple. Genetic analysis of prokaryotic and eukaryotic dna-binding proteins in escherichia coli. Nucleic Acids Res, 26(0305-1048):3700–6, 1998.
  2. Bacterial artificial chromosomes. S. Zhao and M. Stodolsky, editors, Volume 1: Library Construction, Physical Mapping, and Sequencing, volume 255 of Methods in Molecular Biology. Humana Press, 2004.
  3. Bacterial artificial chromosomes. S. Zhao and M. Stodolsky, editors, Volume 2: Functional Studies, volume 256 of Methods in Molecular Biology. Humana Press, 2004.
Personal tools