Multichannel Systems Roboinject Manual - Manual (Page 81)

Appendix
77
 Rubber ball for oocyte transfer pipette
 Glass tool for moving oocytes: Glass Pasteur pipette, sealed over a Bunsen burner,
and melted into a golf-club like shape.
 Petri or cell culture dishes, 100 mm
 Petri dishes, 60 mm
 Beaker, 100 ml
 Razor blade
 Forceps
 Parafilm
 Standard laboratory equipment
6.5.2 Chemicals
Collagenase
For defolliculation:
 Fresh 1.52 mg/ml collagenase in Barth’s solution without Calcium. The concentration has to be
optimized according to the collagenase batch and experimental conditions, see also chapter
"Defolliculation". Do not prepare solutions in advance as collagenase activity may decrease
rapidly even if the solution is stored at -20 °C.
 Collagenase from Cl. histolyticum ca. 0.17 to 0.28 U/mg (according to Wünsch, 1963) lyophilized.
Gentamicin
 Stock solution: 50 μg/ml gentamicin (free base) in Barth’s solution. 1 ml aliquots with 50 mg/ml
gentamicin (free base) are stored at -20 °C.
 Working solution: Dilute 1 ml of gentamicin stock in 1 l Barth's solution.
 Gentamicin sulfate salt, potency approx. 600 μg Gentamicin per mg.
 Alternatively to gentamicin, a mixture of penicillin and streptomycin can also be used to suppress
growth of microorganisms.
Barth’s solution
 pH 7.4 (with NaOH)
 88 mM NaCl
 2.4 mM NaHCO
3
 1 mM KCl
 0.33 mM Ca(NO
3
)2 * 4 H
2
O
 0.41 mM CaCl
2
* 2 H
2
O
 0.82 mM MgSO
4
* 7 H
2
O
 5 mM Tris/HCl