BAC DNA Purification for Transgenic Mouse Production
BAC DNA has been purified successfully for microinjection by three principle methods: Anion Exchange Chromatography (Nucleobond Columns), CsCl Gradient, or Sepharose 4B-CL Chromatography. Any of these methods can be used successfully to purify BAC DNA for pronuclear microinjection when the protocols are followed carefully.
Nucleobond Column Method
CsCl Gradient Method
Sepharose 4B-CL Method
Very Important -> Microinjection Buffer
In our hands, successful BAC preparations require the
polyamine microinjection buffer described below.
We have stored intact BAC DNA at 4C for a year in polyamine buffer.
Buffer composition is 10 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 30 microM spermine, 70 microM spermidine, 100 mM NaCl. This buffer is more likely to produce transgenic mice with intact, unfragmented DNA molecules. See the following references: Schedl A, Larin Z, Montoliu L, Thies E, Kelsey G, Lehrach H, Schutz G. 1993. A method for the generation of YAC transgenic mice by pronuclear microinjection. Nucleic Acids Res 21:4783-4787 and Montoliu L, Bock CT, Schutz G, Zentgraf H. 1995. Visualization of large DNA molecules by electron microscopy with polyamines: application to the analysis of yeast endogenous and artificial chromosomes.. J Mol Biol 246(4):486-492. See also Lluis Montoliu's web site.
1000x Polyamine Stock
30 mM Spermine (Sigma, tetrahydrochloride, #S-1141)
70 mM Spermidine (Sigma, trihydrocholoride, #S-2501)
Dissolve the spermine and spermidine together in autoclaved distilled water, filter sterilize (0.2 micron filters), and store at -20 C. Since the polyamines are very hygroscopic, it is suggested that small quantities (1 gram) should be ordered and then all of it should be prepared at once.
For 50 ml:
10 mM Tris-HCl, pH 7.5 0.5 ml of 1 M Tris-HCl, pH 7.5 (autoclaved)
0.1 mM EDTA, pH 8.0 10 microliters of 0.5 M EDTA, pH 8.0 (autoclaved)
100 mM NaCl 1 ml of 5 M NaCl (autoclaved)
1x Polyamines 50 microliters 1000x Polyamines mix
Autoclaved H2O up to 50 ml
NOTE: Prepare fresh and discard unused microinjection buffer.
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