Recipes/EM

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Contents

Embryo Medium

1 L 5 L 20 L Ingredients Location
(Nalgene Bottle) (Small Carboy) (Large Carboy)
50 ml 250 ml 1,000 ml 20X E.M. Stock Area M, Shelf 3
2 ml 10 ml 40 ml 500x Sodium Bicarbonate Area M, Shelf 3
948 ml 4,740 ml 18,960 ml Distilled Water Area H, Counter

Directions

  1. Mix everything together in a fish safe container. These fish safe containers are marked by red sharpie.






500 X Bicarb aliquots

Weigh out 0.3 g aliquots of sodium bicarbonate into 15 ml culture tubes. Add water and use solution the same day only

10 ml

  • NaHCO3, 0.3 g
  • dH20, q.s. to 10 ml
store at 4 C

For E2 medium, add 50 ml 20X and 2 ml 500x, and q.s. to 1 liter

Embryo media by Don Kane

q.s.- as much as is sufficient
Add dry reagents, then add liquid to bring the total to final amount. Don't measure out final amount of liquid and then add dry reagents.
Latin- quantum sufficit


E medium
  1. NaCl, 13.7 mM
  2. KCl, 0.5 mM
  3. CaCl2*2HOH, 1.3 mM
  4. MgSO4*7HOH, 1.0 mM
  5. KH2PO4, 0.044 mM
  6. Na2HPO4*2HOH, 0.025 mM
  7. NaHCO3, 4.2 mM



E2 medium (the New and Improved embryo medium!)
  1. NaCl, 15.0 mM
  2. KCl, 0.5 mM
  3. CaCl2*2HOH, 1.0 mM
  4. MgSO4*7HOH, 1.0 mM
  5. KH2PO4, 0.15 mM
  6. Na2HPO4*2HOH, 0.050 mM
  7. NaHCO3, 0.70 mM


0.1x MBSH medium
  1. NaCl, 2.9 mM
  2. KCl, 0.10 mM
  3. CaCl2*2HOH, 0.04 mM
  4. MgSO4*7HOH, 0.08 mM
  5. NaHCO3, 0.24 mM
plus
  1. Ca(NO3)2, 0.033mM
  2. Hepes, 1mM



Formulation for 20x E2 medium minus bicarb

1 liter

  1. NaCl, 17.5 g
  2. KCl, 0.75 g
  3. CaCl2*2HOH, 2.9 g
  4. MgSO4*7HOH, 4.9 g
  5. KH2PO4, 0.41 g
  6. Na2HPO4*2HOH, 0.15 g
  7. dH2O, q.s. to 1000 ml



Formulation for 500x bicarb

10 ml

  1. NaHCO3, 0.3 g
  2. dH20, q.s. to 10 ml
  3. store at 4 C
  1. For E2 medium, add 50 ml 20X and 2 ml 500x, and q.s. to 1 liter.



Formulation for E2 medium (large batches)

20 liter

  1. NaCl, 17.5 g
  2. KCl, 0.75 g
  3. CaCl2*2HOH, 2.9 g
  4. MgSO4*7HOH, 4.9 g
  5. KH2PO4, 0.41 g
  6. Na2HPO4*2HOH, 0.15 g
  7. NaHCO3, 1.2 g
  1. dH2O, q.s. to 20 l
  2. should be pH 7.0 to 7.5
  3. Filter sterilize into sterile 500 ml bottles.
  4. Refrigerate.



For quick 100% ad libed Hanks, add 0.8 g NaCl and 0.04 g KCl to 100 ml of E2 media. This works for collecting sperm.



Formulation for Pen-strep addition:

100 ml

  1. Streptomycin*, 10 g
  2. Penicillin, 1690U/mg **, 6 g
  3. dH2O, q.s. to 100 ml
Filter sterilize. Freeze in 1 ml aliquots. Dilute 1:1000.
This dilution gives 0.1 mg/ml strep and ~100 U/ml pen. This is the conc that we (Robert Ho and Don) used for transplantations.
In E2 medium, is stable a week and room temp; it is not stable for a month.


For some mysterious reason, kills embryos in selmarin even when diluted tenfold.


  • Streptomycin Sulfate. Merck #10 177 MW = 1457.39 (C21H39N7O12)2*(H2SO4)3
  • Penicillin G (Benzylpenicillin Sodium Salt). Sigma P3032 1690Units/mg FW=356.4 C16H17N2O4SNa
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