TRANSGENIC FACILITY
Samuel Lunenfeld Research Institute, Mount Sinai Hospital
600 University Ave., Toronto, Ontario, M5G 1X5
416-586-8382
MilliQ, triple-distilled or purchased ultra pure water
(Gibco, BDH, Sigma) is used to prepare M2, M16 and KSOM media .
Water that has been purified by a combination of reverse osmosis
and MilliQ filtration contains minimal contaminants. Only if the conductivity
is 18 megOhms or more the is the water purification system satisfactory.
Prolonged storage of water is not advised.
Clean glass bottles that have never been exposed to detergent
or organic solvents, and have been thoroughly rinsed
(at least 6 times in distilled water) or plastic tissue culture
flasks are used to collect water and prepare stocks and media.
Plastic ware : tissue culture bottles, flasks, polypropylene
tubes, disposable pipettes are preferable.
0.2 micron Nalgene filter unit is used for filter sterilization.
Some filter units might contain something toxic to embryos.
First 10 - 20 ml of filtered solution are disposed or some
PBS is filtered through the unit and disposed before filtering media.
All chemicals should be of the highest grade possible. It
is often necessary to test several batches of particular component (e.g.
bovine serum albumin and mineral oil). Some embryo tested chemicals
might be ordered from Sigma. Chemicals used to prepare media should
be reserved only for this purpose. 1 N NaOH, 1 N HCl are made
according to the above requirements for water and containers and should
be replaced regularly. The use of separate pH meter electrode is suggested.
The concentrated stock solutions can be stored at -75 C for
6 months (or -20 C for shorter time). The final working medium is filter
sterilized and stored into aliquots at +4 C for 2-3 weeks. It
is best to replace these every other week
(Modified from "
Manipulating the Mouse Embryo" A Laboratory Manual,
Hogan B, F. Constantini,
E. Lacy, 1986, Cold Spring Harbor Laboratory).
| COMPONENT | g / 100 ml | |
| STOCK A (10X) | NaCl | 5.534 |
| KCl | 0.356 | |
| KH2PO4 | 0.162 | |
| MgSO4 x 7 H2O | 0.293 | |
| Sodium Lactate | 2.610 or 4.349 g of 60% syrup | |
| Glucose | 1.000 | |
| Penicillin G | 0.060 | |
| Streptomycin | 0.050 | |
| STOCK B (10X) | NaHCO3 | 2.101 |
| Phenol Red | 0.001 | |
| STOCK E (10X) | HEPES | 5.958 |
| Phenol Red | 0.001 | |
| COMPONENT | g / 10 ml | |
| STOCK C (100X) | Sodium Pyruvate | 0.036 |
| STOCK D (100X) | CaCl2 x 2 H2O | 0.252 |
Weigh solids into media bottles and add appropriate quantity
of water. If Sodium Lactate syrup is used (stock A), the boat should be
rinsed well and the wash added to the flask. For stock E add half of water
volume, then adjust the pH to 7.4 with 1 N NaOH. Make up to volume using
cylinder. Filter stocks through Millipore filter, aliquot into sterile tubes.
Preparation of 100 ml of M2 and 100 ml of M16 from concentrated
stocks:
| STOCK | M2 (ml) | M16 (ml) |
| A (10X) | 10.0 | 10.0 |
| B (10X) | 1.6 | 10.0 |
| C (100X) | 1.0 | 1.0 |
| D (100X) | 1.0 | 1.0 |
| E (10X) | 8.4 | - |
| WATER | 78.0 | 78.0 |
| BSA (Bovine Serum Albumin, Sigma A4378 | 400 mg | 400 mg |
In February 1998 we replaced the M16 medium with KSOM in all our experiment. Our preliminary data show improvement in all the applications.