Luria Broth LB
(prepare with dd H2O, sterlilized by autoclaving)
1% w/v bacto-tryptone
0.5% w/v yeast extract
1% w/v NaCl
pH adjusted to 7.0 using NaOH 0,6 N
Solution 1 ( Cell Resuspension Solution)
50 mM Glucose (4.5 mL; stock 20%)
25 mM Tris pH 8 (2.5 mL; stock 1 M)
10 mM EDTA (10 mL; stock 0.1 M)
Solution 2 ( Cell Lysis Solution, Fresh)
2 M NaOH 1 mL
10 % SDS 1 mL
Dd H2O 8 mL
Solution 3 ( Neutralisation Solution)
5 M potassium acetate 60 mL
Glacial cetic acid 11.5 mL
H2O 28.5 mL
2X TSS (Competent cells media, TSS methods)
TSS=LB + 10% PEG + 5% DMSO + 20-50 mM MgCl2
pH=6.5
Glycerol-CaCl2 Solution (competent cells media, CaCl2 methods)
22.5 mL 0.4 M CaCl2
28.1 mL 80% Glycerol
99.4 mL H2O
TAE 50X/L
242 g Tris Base
57.1 mL Glacial acetic acid
100 mL 0.5 M EDTA pH 8
TBE 5X/L
54 g Tris Base
27.5 g Boric Acid
20 mL 0.5 M EDTA pH 8
EtBr
Stock Solution 10 mg/mL ( 1 tablet / 1 mL H2O)
Working Solution 0.5 ug/mL (250 mL H2O)
Double Detergent Lysis Bufer (100 mL)
50 mM Tris-HCl pH 8
150 mM NaCl
0.1% SDS
1% IGEPAL
SDS-PAGE Gel
12% resolving gel (1 gel)
3.4 mL H2O
2.5 mL 1.5M Tris-HCl pH 8.
50 uL SDS 20%
4 mL Acrylamida/bis
50 uL APS 10%
5 uL TEMED
12% stacking gel (2 gel)
3.075 mL H2O
1.25 mL Tris-HCl pH 6.8
25 uL SDS 20%
0.67 mL Acrylamida/bis
25 uL APS 10%
5 uL TEMED
8% Resolving gel (2 gel)
9.3 mL H2O
5.0 mL Tris-HCl pH 8.8
0.1 mL SDS 20%
5.3 mL Acrylamida/bis
0.2 mL APS 10%
0.012 mL TEMED
8% Stacking gel = 12% Stacking gel
Low Toxicity Staining Solution
0.25 g coomassie blue R250
100 mL ethanol
100 mL water
Stir 1 hour
+ 25 mL Acetic Acid
+water until 250 mL
Stored at room temperature (Alumunium foiled bottle)
Destaining Solution
400 mL ethanol
100 mL acetic acid
500 mL water
Stored at room temperature