Team:Bielefeld-CeBiTec/Media

iGEM Bielefeld 2015

Media

LB

For 1 liter LB:

20 g LB powder
Fill the bottle with deionized H₂O

For 1 liter LB-plates:

18 g LB powder
16 g Select Agar
Fill the bottle with deionized H₂O

SOC

Add the following components for 900 ml of distilled H₂O:

20 g Trypton
5 g Bacto Yeast Extract
2 ml of 5 M NaCl
2.5 ml of 1 M KCl
10 ml of 1 M MgCl2
10 ml of 1 M MgSO4
20 ml of 1 M glucose

Buffers

50X TAE Stock Solution

For each litre of solution:

242 g Tris Base (MW=121.1)
57.1 ml Glacial Acetic Acid
100 ml 0.5 M EDTA

mix Tris with stir bar to dissolve in about 600 ml of ddH₂O.
add the EDTA and Acetic Acid.
bring final volume to 1 l with ddH20.
store at room temperature.

Note: Final (1x) working concentration:

0.04 M Tris - Acetate
0.001 M EDTA

Lysis and binding buffer for purification of lacI

For each 200 ml of solution:

5.844 g NaCl
0.272 g imidazole
5.8 ml of 86 % glycerol
0.406 g MgCl₂
0.2 g lysozyme

Add all these ingredients to a 50 mM sodium phosphate buffer (pH 8.0). Prior to use add DTT to a final concentration of 1mM and tween 20 to a final concentration of 0.1 %.
store at room temperature.

Note: Final (1x) working concentration:

50 mM Sodiumphosphate
500 mM NaCl
20 mM imidazole
2.5 % glxcerol
1 mM DTT
10 mM MgCl₂
0.1 % tween 20
1 mg/ml lysozyme

Equilibration buffer for lacI purification

For each litre of solution:

29.22g NaCl
1.36 g imidazole
29 ml of 86 % glycerol

Add all these ingredients to a 50 mM sodium phosphate buffer (pH 8.0). Prior to use add DTT to a final concentration of 1mM.
store at room temperature.

Note: Final (1x) working concentration:

50 mM sodium phosphate
500 mM NaCl
20 mM imidazole
2.5 % glycerol
1 mM DTT

Elution buffer for purification of lacI

For each litre of solution:

17.53 g NaCl
13.6 g imidazole

Add all these ingredients to a 20 mM sodium phosphate buffer (pH 7.4).
store at room temperature.

Note: Final (1x) working concentration:

20 mM sodium phosphate
300 mM NaCl
200 mM imidazole

Buffer for dialysis of lacI

For each litre of solution:

50 g glucose

Add the glucose to a 200 mM potassium phosphate buffer (pH 7.6). Prior to usage add DTT to a final concentration of 1 mM
store at room temperature.

Note: Final (1x) working concentration:

200 mM potassium phosphate
5 % glucose
1 mM DTT

Buffers for CFPS

Stock solutions for CFPS

1.5 M Spermidine and 1 M putrescine solutions are prepared according to supporting material from Yang et al. 2012a. Briefly, a total of 23 mL of autoclaved MilliQ water is added in small steps to a 5 g bottle of spermidine. After each step, the bottle is mixed vigorously and the liquid is poured into a beaker where it is continously stirred. After rinsing out the whole bottle, the homogenous solution is aliquoted, flash freezed and stored at -80 °C. The procedure is similar for putrescine, though volumes differ.