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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
or
CsCl Gradient Method
or
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.


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Preparation of Microinjection 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.

Microinjection Buffer

     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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