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Reagents for Generation of Targeted Clones
MEF Media (0.2mM nitrocellulose filtered)
440 ml DMEM
50 ml FCS (Hyclone, Heat Inactivated)
5 ml L-Glutamine (200mM) (Gibco #ESG1107)
5 ml Non-Essential Amino Acids (10mM each) (Gibco #11140-050)
Add 0.5 ml Penicillin-Streptomycin (P/S) 1000X only if making a new prep of MEFs from mouse embryos, otherwise do not add the P/S to your media. Adjust the amount of DMEM accordingly if adding the P/S. Make this media fresh every two weeks.
ES Media (0.2mM nitrocellulose filtered)
405 ml DMEM
85 ml FCS (Hyclone Heat Inactivated)
5 ml L-Glutamine (200 mM)
5 ml Non-Essential Amino Acids (10 mM each) (Gibco #11140-050)
2.5 ml 2M Hepes pH7.2
50 ul LIF (1000 units/ml final) (Chemicon #ESG1107)
5 ul 2-Mercaptoethanol (Sigma #M7522)
Make this media fresh every week.
Selection Media (0.2mM nitrocellulose filtered)
500 ml ES Media above
Final Concentration of 300 ug/ml Active Geneticin (G418) (Sigma #G9516)
To arrive at the correct amount by weight: Take the activity provided on your Geneticin bottle and perform the following calculation:
eg. 706 ug active geneticin = 300 ug X=425 ug
__________________ _____
mg X
425 ug X 500 ml. = 212.5 mg by weight/500 ml.
Dilute the Geneticin in 3 ml. PBS and sterile filter with an
0.2 uM Nitrocellulose filter and add to the ESmedia.
2 X Cell Freezing Media (0.2mM nitrocellulose filtered)
30 ml ES media
10 ml FCS (Hyclone Heat Inactivated)
10 ml DMSO (Sigma #D8779)
For freezing 120 clones you will need at least 60 ml. prepared.
Transfection Buffer (1 X Hebs) (0.2mM nitrocellulose filtered)
20mM Hepes pH 7.05
137mM NaCl
5mM KCl
0.7mM Na2HP04 (dibasic)
6mM Dextrose
Fetal Calf Serum (Hyclone #A-1119-L)
Heat inactivated at 56oC for 30 minutes. Lot tested according to Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Edited by E.J. Robertson, Chapter 4, Section 2.3.1 Serum testing.
Supplies and Potential Source List
Dulbecco's Modified Eagle's Medium (DMEM) 1X, high glucose, with L-glutamine, without sodium pyruvate. Washington University Tissue Culture Center or Gibco BRL. Cat.# 11965-084
L-Glutamine (200mM) 100X Washington University Tissue Culture Center or Gibco BRL. Cat.# 25030-081
Non-Essential Amino Acids (10 mM each) 100X Washington University Tissue Culture Center or Gibco BRL. Cat.# 11140-050
Penicillin-Streptomycin (10000 units/10000ug/ml) 1000X Washington University Tissue Culture Center or Gibco BRL. Cat.# 15140-122
Trypsin/EDTA (0.05%/0.02%) Washington University Tissue Culture Center or Gibco BRL. Cat.# 25300-047
Hepes Buffer 2.5M Washington University Tissue Culture Center
Leukaemia Inhibitory Factor Chemicon (800) 437-7500 Cat.# ESG1107
2-Mercaptoethanol (2ME) Sigma Chemical Co. Cat.# M7522
Dimethyl Sulfoxide (DMSO) Sigma Chemical Co. Cat.# D8779
Geneticin Sigma Chemical Co. Cat.# G9516
Hyclone Fetal Calf Serum Hyclone Laboratory Cat.# A-1119-L
Lot test-see EJ Robertson Tertocarcinomas and Embryonic Stem Cells
Heat inactivate before use @ 56oC for 30 minutes.
Flatpack chambers 1.83 mm gap BTX Part#485
Electroporation cuvettes 2mm gap BTX Cat.# 847
Cryo-tube 1.8ml S1 Nunc Fisher Scientific Cat.# 377267
Murine Embryonic Fibroblasts Specialty Media (800) 543-6029
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