Microinjection buffer for BAC/YAC DNA

EDTA (0.5 M, pH 8.0)

NaCl (5 M)

Spermidine (Sigma, trihydrochloride)

Spermine (Sigma, tetrahydrochloride)

Tris-Cl (1 M, pH 7.5)

1. For the 1000x polyamine mix, dissolve the spermine and spermidine together in sterile H₂O so that the end concentration is 30 mM spermine and 70 mM spermidine.

2. Filter sterilize (0.22 micron) the mixture and store at -20°C.

3. For the basic injection buffer, add 0.5 ml of 1 M Tris-Cl to a plastic, disposable 50-ml Falcon tube.

4. Add 10 μl of 0.5 M EDTA.

5. Add 1 ml of 5 M NaCl.

6. Add sterile H₂O up to 50 ml.

7. Aliquot, filter sterilize, and store at 4°C.

8. To prepare reay-to-use injection buffer, add 50 μl of polyamine mix to 50 ml of basic injection buffer. Mix and use directly.