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Tables of Media Composition, 1M DMSO, DAP213, 0.25M sucrose, mHTF and KSOM

 Freezing Medium and Culture Medium for Mouse Reproductive Engineering

  Mouse General Freezing and Culture Medium

   Reproductive Engineering Manuals

1M DMSO

Reagent  Name mg/100ml Vendor Catalog Number
NaCl 555 Sigma S 5886
KCl 18.5 Sigma P 5405
KH2PO4 4.75 Sigma P 5655
MgSO4 7H2O 4.95 Sigma M 7774
CaCl2 2H2O 25 Sigma C 7902
NaHCO3 210 Sigma S 5761
Glucose 3.6 Sigma G 6152
Na-Pyruvate 2.2 Sigma P 4562
DL-Lactic Acid, sodium salt 0.174ml Sigma L 1375
10mM EDTA 100µl Sigma E-6635
Streptomycin 5 Sigma S 9137
Penicillin 6.3 Sigma P 7794
0.5% phenol red 0.1ml Sigma P 0290
L-Glutamine 14.6 Sigma G 8540
MEM Essential Amino Acids 1ml Sigma 11130-051
MEM Non-essential AA 0.5ml Sigma M 7145
BSA 100 Sigma A-4378

Reference
Nakao K., Nakagata N., and Katsuki M. 1997. Simple and effcient procedure for cryopreservation of mouse embryos by simple vitrification. Exp. Anim. 46: 231-234.

DAP213

2M DMSO, 1M Acetamide, 3M Propylene Glycol in PB1

PB1

Reagent  Name mg/100ml Vendor Catalog Number
NaCl 800.0 Sigma S5886
KCl 20.0 Sigma P5405
CaCl2 12.0 Sigma C5670
KH2PO4
20.0/td> Sigma P5655
MgCl2 6H2O
10.0 Sigma M2393
Na2HPO4 115.0 Sigma S5136
Na-Pyruvate 3.6 Sigma P4562
Glucose 100.0 Sigma G6152
Penicillin 7.5 P4687 L 1375
Streptomycin 5.0 Sigma S1277
BSA 300.0 Sigma A4378


DAP213 : Solution A and Solution B are first prepared and are each fully dissolved. Equal volumes of A and B are then mixed to form DAP213. Caution: The solution may become cloudy when DMSO is added.

Solution A

Reagent  Name mL (Final volume 10mL) Vendor Catalog Number
PB1 2.3088 - -
DMSO 3.1252 Sigma D2650
Propylene glycol (PG) 4.556 Sigma P1009

Solution B

Reagent  Name mg/10mL in PB1 Vendor Catalog Number
Acetamide (AA) 1181.4 Sigma A0500

Reference
Nakao K., Nakagata N., and Katsuki M. 1997. Simple and effcient procedure for cryopreservation of mouse embryos by simple vitrification. Exp. Anim. 46: 231-234.

0.25M Sucrose

PB1

Reagent  Name mg/100ml Vendor Catalog Number
NaCl 800.0 Sigma S 5886
KCl 20.0 Sigma P-5405
CaCl2 12.0 Sigma C 5670
KH2PO4 20.0 Sigma P5655
MgCl2 6H2O 10.0 Sigma M 2393
Na2HPO4 3.6 Sigma S 5136
Na-Pyruvate 3.6 Sigma P-4562
Glucose 100.0 Sigma G-6152
Penicillin 7.5 Sigma P-4687
Streptomycin 5.0 Sigma S-1277
BSA 300.0 Sigma A-4378

0.25M Sucrose in PB1

Reagent  Name mg/20ml in PB1 Vendor Catalog Number
Sucrose 1711.5 Sigma S-1888

Reference
Nakao K., Nakagata N., and Katsuki M. 1997. Simple and effcient procedure for cryopreservation of mouse embryos by simple vitrification. Exp. Anim. 46: 231-234.

mHTF

Reagent Name mg/100ml Vendor Catalog Number
NaCl 593.8 Sigma S 5886
KCl 35.0 Sigma P5405
MgSO4 7H2O 4.9 Sigma M7774
KH2PO4 5.4 Sigma P5655
CaCl2 57.0 Sigma C5670
NaHCO3 210.0 Sigma S5761
Glucose 50.0 Sigma G6152
Na-lactate (mL)* 0.34mL Sigma L7900
Na-Pyruvate 3.7 Sigma P4562
Penicillin G 7.5 Sigma P4687
Streptomycin 5.0 Sigma S1277
BSA
(Albumin, Bovine serum, Fraction V, Fatty Acid-Free)
400 MERCK/CALBIOCHEM 126575
0.5% phenol red (mL) 0.04mL Sigma P0290

* Assay; 70%

Reference
Kito S., Hayao T., Noguchi-Kawasaki Y., Ohta Y., Hideki U., and Tateno S. 2004. Improved in vitro fertilization and development by use of modified human tubal fluid and applicability of pronucleate embryos for cryopreservation by rapid freezing in inbred mice. Comp. Med. 54(5): 564-570.

KSOM

Reagent  Name mg/100ml Vendor Catalog Number
NaCl 555 Sigma S 5886
KCl 18.5 Sigma P 5405
KH2PO4 4.75 Sigma P 5655
MgSO4 7H2O 4.95 Sigma M 7774
CaCl2 2H2O 25 Sigma C 7902
NaHCO3 210 Sigma S 5761
Glucose 3.6 Sigma G 6152
Na-Pyruvate 2.2 Sigma P 4562
DL-Lactic Acid, sodium salt 0.174ml Sigma L 1375
10mM EDTA 100µl Sigma E-6635
Streptomycin 5 Sigma S 9137
Penicillin 6.3 Sigma P 7794
0.5% phenol red 0.1ml Sigma P 0290
L-Glutamine 14.6 Sigma G 8540
MEM Essential Amino Acids 1ml Gibco 11130-051
MEM Non-essential AA 0.5ml Sigma M 7145
BSA 100 Sigma A-4378

Reference
Lawitts J.A., and Biggers J.D. 1993. Culture of preimplantation embryos. Methods Enzymol. 225:153-164.

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.