Millipore Technical Publications |
 | Protocol - MCPROTO038 | |
Protocol: Reagents | ||
| Lit No: | MCPROTO038 | |
| Year: | 2007 | |
Reagents
Leishmann’s Stain
(BDH Product #35022)
Make to 0.2% w/v solution in methanol.
Gurrs Buffer Tablets pH6.8
(BDH Product #33193)
Add 1 mL of 0.2% Leishmann’s stain solution to 5 mL of pH 6.8 buffer. This is sufficient for 2-3 slides. This must be made freshly for a few slides at a time, as it will precipitate.
Fibroblast Media:
500mL knockout DMEM (GibcoBRL, Life Technologies)
50mL FBS (Trace Biosciences Pty Ltd.)
5mL 100x pen/strep
5mL 100x glutamine
3.5mL mercaptoethanol
Store at 4°C, in foil for up to 4 weeks.
Add fresh glutamine after this time.
Freezing Media:
80% FBS (Trace Biosciences Pty Ltd.)
10% fibroblast media
10% DMSO
Store stock DMSO wrapped in foil at 4°C.
10X PBS:
1.44g KH2PO4
7.95g Na2HPO4
90g NaCl
Make up to 1L with water suitable for tissue culture. Freeze in smaller aliquots or autoclave. Dilute 10X stock to 1X with water and autoclave for use. Alternatively use PBS tablets in tissue culture suitable water (1 tablet per 100mL water).
ES Media (with Fetal Bovine Serum):
200mL fibroblast media
2mL non-essential amino acids
ESGRO® (1000 units/mL is recommended).
For selection media add:
150-300mg/mL G418 (Geneticin)
ES Media with Serum Replacement:
500mL knockout DMEM (GibcoBRL, Life Technologies)
90mL knockout serum replacement (15%) (Knockout SR, GibcoBRL, Life Technologies)
6mL 100x non-essential amino acids (Trace Biosciences Pty. Ltd)
6mL 100x pen/strep
6mL 100x glutamine
4.6mL mercaptoethanol
ESGRO® (1000 units/mL is recommended).
NB. The serum replacement media will not inactivate trypsin. Spin down to remove trypsin when splitting cells or use soybean trypsin inhibitor.
MT-PBS:
0.227g Na2HPO4
0.0625g NaH2PO4.2H20
0.87g NaCl
Make up to 100mL and filter sterilize.
Lysis Buffer:
2mL 1m Tris pH 8.5
0.2mL 0.5M EDTA
0.2mL 20% SDS
0.8mL 5M NaCl
100mL 20mg/mL proteinase K
100mL 10mg/mL RNaseA
16.8m: MQW