Media and Solutions

Medium / Solution Compounds Notes

Agarose-Gel 1% Agarose in TAE-Buffer

Boil 2 g Agarose in 200 ml TAE- Buffer in the mikrowave oven respct. Boil on a hotplate while stiring until the solution is clear

Blocking Solutions 4% BSA in 1x PBS/0.01% Tween 2% BSA in 1x PBS/0.01% Tween 4 g BSA → ad 100 ml mit 1x PBS/0.01% Tween 20 ml 4% BSA in 1x PBS/0.01% Tween + 20 ml 1x PBS/0.01% Tween

Complete Medium with 200 nM NCS

Dependent on the celltype respective Complete Medium, NCS 500 µM 10 ml Complete Medium + 4 µl NCS DAPI-Working Solution 0.2 µg/ ml DAPI-Solution 10 µg/ml 1x PBS oder 2x SSC Correspends to a dilution of 1:50 _→ e.g. 5 µl DAPI-Soluton + 245 µl 1x PBS; use 2x SSC instead of 1x PBS for FISH

DAPI-Solution 10 µg/ml DAPI-Stock Solution 500 µg/ml _{1x PBS} 20 µl DAPI-Stock Solution + _{980 µl 1x PBS} DAPI-Stock Solution 500 µg/ml DAPI H₂O_bidest. 10 mg DAPI + 20 ml H₂O_bidest.

DNA-Buffer 100 mM EDTA 200 mM Tris

2 ml 0.5 M EDTA + 2 ml 1 M Tris/HCl pH 7.8 or 8.0 + 6 ml H₂O_bidest.

Dulbecco’s MEM complete medium

Dulbecco’s MEM Medium, FCS, Penicillin/Streptomycin

450 ml Dulbecco’s MEM Medium + 50 ml FCS + 5 ml Penicillin/Streptomycin

EDTA 0.5 M, pH 8.0 EDTA, H₂O_bidest.

1.86 g EDTA → ad 8 ml

H₂O _bidest.; adjust with 1 N NaOH to pH 8.0

Ethanol 70% Ethanol technical, H₂O_dest 75 ml Ethanol + 175 ml H₂O_dest.

Ethanol 90% Ethanol technical, H₂O_dest. 25 ml Ethanol + 225 ml H₂O_dest.

Ethidium Bromide 1 mg/ml Ethidium-Bromide, H₂O_bidest. 10 mg Ethidium-Bromide + 10 _{ml H}

2Obidest.

Ethidium Bromide Bath 1x TAE, 1 mg/ml Ethidium-_Bromide 600 ml 1x TAE + 6 drops 1 mg/_{ml Ethidium-Bromide} Freezing medium 10% DMSO in FCS 9 ml FCS + 1 ml DMSO

Glycerin-Solution 20% Glycerin, 1x PBS 50 ml Glycerin + 200 ml 1x PBS 119.1 g HEPES _→ ad 500 ml

Medium / Solution Compounds Notes

HEPES / NaOH pH 7.4 100 mM 1 M HEPES / NaOH pH 7.0, _H

2Odest.

5 ml 1 M HEPES / NaOH pH 7.0 + 45 ml H₂O_dest.; adjust with 1 N NaOH to pH 7.4

“Holland”-Medium

Dulbecco’s MEM Medium, Ham’s F10 Medium, FCS, Peni- cillin/Streptomycin

22.5 ml Dulbecco’s MEM Medium + 22.5 ml Ham’s F10 Medium + 5 ml FCS (= 10%) + 500 µl Penicillin/Streptomycin

“Holland”-Medium w/o Phe- nolred

Dulbecco’s MEM Medium w/o Phenolred, Ham’s F-10 Medium w/o Phenolred, FCS, Penicillin/ Streptomycin, Glutamine 200 mM

22.5 ml Dulbecco’s MEM Medium w/o Phenolrot + 22 ml Ham’s F10 Medium w/o Phenolred + 400 µl Glutamine + 5 ml FCS + 500 µl Penicillin/ Streptomycin

“Holland”-Medium w/o Phe- nolred with 100 nM Trolox

Holland-Medium w/o Phe- nolred, 100 mM Trolox

10 ml Holland-Medium w/o Phenolred + 10 µl 100 mM Trolox

Hypotonic-Solution (0.56% KCl) KCl, H₂O_dest. 1.4 g KCl → ad 250 ml H₂O_dest.

Hypotonic Solution for Multiporator by Eppendorf

λ HindIII-Marker λ_H HindIII, Loading Dye 6x,

2Obidest.

100 µl λ HindIII + 100 µl 6x Loading Dye + 50 µl H₂O_bidest

Methanol/Acetic Acid Methanol/Acetic Acid 3:1 (V:V) 150 ml Methanol + 50 ml Acetic _Acid

NaOH 1 N NaOH, H₂O_bidest. 20 g NaOH _H → ad 500 ml

2O bidest.

Paraformaldehyde 4% respct. 1% PFA in 1x PBS

Solve 4 g respct. 1 g PFA in 100 ml 1x PBS completely while stiring and heating. Cool down before use.

PBS-Buffer 1x

For Cell-Culture usage: 20x PBS (→ 140 mM NaCl, 2.7 mM KCl, 6.5 mM Na₂HPO₄, 1.5 mM KH₂PO₄), H₂O_bidest

for Lab usage: contains 0.04% Sodium Azide

25 ml 20x PBS + 475 ml H₂O _bidest.; → autoclave

1 l 1x PBS + 1 small spatula-tip Sodium Azide

PBS-Buffer 1x / 0.01% Tween 1x PBS, Tween 20 50 µl Tween 20 + 500 ml 1x _PBS

PBS-Buffer 20x, pH 7.4 2.8 M NaCl, 54 mM KCl, 130 _{mM Na} 2HPO4, 30 mM KH2PO4 320 g NaCl + 8 g KCl + 57.6 g Na₂HPO₄ + 9.6 g KH₂PO₄→ ad 2 l H₂O_bidest., adjust pH to 7.4 with HCl

Pepsin Stock Solution Pepsin, H₂O_bidest. 1 g Pepsin + 10 ml H₂O_bidest.

Poly-L-Lysin Working Solution

1.25 mg/ml 10 mg/ml Poly-L-Lysin-Stock Solution, H₂O_bidest.

1 ml 10 mg/ml Poly-L-Lysin- Stock Solution + 7 ml H₂O_bidest.

Poly-L-Lysin Stock Solution 10 mg/ml

Poly-L-Lysin-Hydrobromide, H₂O_bidest.

100 mg Poly-L-Lysin-Hydrobro- mide + 10 ml H₂O_bidest.

RPMI (Rosswell Park Memo- rial Institute)1640 Complete Medium

RPMI 1640 Medium, FCS, Peni- cillin/Streptomycin

450 ml RPMI Medium + 50 ml FCS + 5 ml Penicillin/Strepto- mycin

TAE-Buffer 1x 50x TAE (_{tate, 1 mM EDTA), H}→ 40 mM Tris-Ace-

2Obidest.

40 ml 50x TAE + 1960 ml H₂O _bidest.

Medium / Solution Compounds Notes

TAE-Buffer 50x, pH 8,0 2 M Tris-Acetate, 0.05 M EDTA

242 g Tris +18.6 g EDTA + 57.1 ml glacial acetic acid → ad 1 l H₂O_bidest.

TE-Buffer 10 mM Tris, 1 mM EDTA

20 µl 1 M Tris/HCl pH 7.8 or 8.0 + 4 µl 0,5 M EDTA + 1978 µl H₂O_bidest.

TE-Buffer ohne EDTA 10 mM Tris 20 µl 1 M Tris/HCl pH 7.8 or 8.0 _{+ 1980 µl H}

2Obidest.

Tris-HCl 1 M, pH 7.5 Tris, H₂O_bidest.

60.6 g Tris → ad 500 ml H₂O _bidest.; adjust with HCl at pH 7.5

Triton-X-100 0,05% 1x PBS, Triton-X-100 50 ml 0.5% Triton-X-100 + 450 _{ml 1x PBS}

Triton-X-100 0.5% 1x PBS, Triton-X-100 2.5 ml Triton-X-100 + 500 ml 1x _PBS

Trolox 100 mM Trolox, 1 N NaOH, 1 N HCl, _H

2Obidest.

0.25 g Trolox → ad 9.8 ml H₂O _bidest. + 100 µl 1 N NaOH, vortex, until all of the Trolox is dissolved (clear, yellow); neu- tralize with 100 µl 1 N HCl

Trypsin/EDTA 1x Trypsin/EDTA 10x, H₂O_bidest. 50 ml 10x Trypsin/EDTA + 450 _{ml H}

2Obidest.