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Experimental Protocols
Embryo Medium
Embryonic Ganglia
Luria Broth
50x TAE
Antibody Staining
Pond Water
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Leech Embryo Culture Medium
50 ml embryo culture medium
L15 50 ml
L-glutamine 5 mg
glucose 55 mg
KCL 30 mg
gentamicin 2.5 mg
ITS+ 0.5 ml
antibiotic/antimycotic 1 ml
fresh Vitamin C 3 mg (0.3 mM), adjust pH
optional:
EGF 5 microgram
SBTI 5 mg
CMK 0.1 mM
Sterilize by filtration through Sterile Millex-GV, 0.22 .m filter units (the yellow filter has a lower absorbance)
Our way of making it
1. Combine:
L15 50 ml
gentamicin 2.5 mg
ITS+ 0.5 ml
antibiotic/antimycotic 1ml
2. Add frozen stocks of:
glucose/KCL/glutamine (100x)
vitamin C (100x)
EGF (1000x)
100 aliquots (0.5 ml) of glucose/KCl/L-glutamine (100x), for 50 ml L15, store at -20C
To 50 ml of water, add glucose (5.5g), KCl (3g), L-glutamine (0.5g)
100 aliquots (0.5 ml) of vitamin C (100x), for 50 ml L15, store at -20C
To 40 ml water, add 264 mg Vitamin C and 240 mg HEPES. Adjust pH to about 7.35. Bring the volume up to 50 ml.
The 0.2 mM HEPES makes it easier to adjust the pH of the Vitamin C solution.
Instead of Vitamin C, you can use 0.2 mM L-Ascorbic Acid 2-phosphate because it is more stable. You may have to adjust the pH.
20 aliquots (50 microliter) EGF (1000x), for 50 ml L15, store at -70 C
Dissolve 100 microgram of EGF in 1 ml water. Freeze as 50 microliter aliquots (for 50 ml L15).
Ordering information
L-Ascorbic Acid 2-phosphate (Sigma A-8960, 5 mg)
antibiotic/antimycotic: Sigma A-9909, 100 ml, freeze in 1 ml aliquots
SBTI Sigma T-9003
CMK: Bachem N-1505.0005. Dissolve the 5 mg of CMK into 67 microliter methanol (100 mM CMK). Use in a 1:1000 dilution.
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