Team:Bielefeld-CeBiTec/Media

iGEM Bielefeld 2015

Media & Buffers

All of our favorite recipes.

Media

LB

For 1 liter LB:

20 g LB powder
Fill the bottle with deionized H₂O

For 1 L 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

2xYT+P

For 1 L, combine

16 g tryptone
10 g yeast extract
5 g NaCl
22 mL KH₂PO₄
40 mL K₂HPO₄

dissolve in water and titrate to pH = 7.0 with NaOH, autoclave

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/ wash buffer for PRIA

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

Binding buffer for PRIA

10 mM TRIS-HCl
Adjust pH 8 with hydrogen chloride.
Add 1 mM DTT to the buffer.
Add KCl or NaCl as needed.
For the optimal binding buffer 500 mM KCl is added.

HEPES (storage of proteins)

50 mM HEPES
Adjust pH 7.2 with hydrogen chloride.

EMSA running buffer

20 mM Na₂HPO₄, pH 8

EMSA buffer for reactions

100 mM sodium phosphate buffer, pH 8.0
375 mM KCl
25% glycine

Buffer for CBD-purification

20 mM Na₂HPO₄*2 H₂
1.5 M NaCl
1 mM EDTA
Dissolve all substances in water.
Adjust pH 8 with phosphoric acid.

Antibody stripping buffer

0.2 M glycine
0.05% Tween 20
Adjust pH 2.5 with hydrogen chloride.

Blocking solution (DNA immobilization)

0.1 M TRIS-HCl, pH 9.0
50 mM ethanolamine

Blocking solution (unspecific protein interaction)

1x PBS
3% milk powder solution

Buffer for annealing of oligonucleotides (PRIA)

50 mM sodium phosphate, pH 8.5

Buffers and stock solutions for CFPS

Buffers

S30A buffer is prepared according to Sun et al. 2013 and contains
- 14 mM Mg-glutamate
- 60 mM K-glutamate
- 50 mM TRIS
at pH = 7.7 (with acetic acid), autoclaved, stored at 4 °C. Right before use, DTT is added to 2 mM final concentration.
S30 buffer (buffer A from Kwon and Jewett 2015) with lower TRIS concentration
- 14 mM Mg-glutamate
- 60 mM K-glutamate
- 10 mM TRIS
at pH = 8.2 (with acetic acid), autoclaved, stored at 4 °C. Right before use, DTT is added to 2 mM final concentration.

Stock solutions

1.5 M Spermidine and 1 M putrescine solutions are prepared according to supporting material from Yang et al. 2012a. Briefly, a total of 4.59 mL of autoclaved MilliQ water is added in small steps to a 1 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.
NTP mixture is prepared from ATP, CTP, GTP and UTP stock solutions (100 mM) from Thermo Scientific. 20x NTP premix contains 30 mM of ATP and GTP, 18 mM CTP and UTP.
330 mM Phosphoenolpyruvate (PEP) stock solution is prepared and pH is adjusted with 5 M KOH according to supporting material from Yang et al. 2012a

Other

10x running buffer for SDS-PAGE

250 mM TRIS-HCl
1.92 M Glycine
1 % (w/v) SDS
pH 8.3

Staining solution for SDS-PAGE

2 g/L Coomassie Brilliant Blue R250
0.5 g/L Coomassie Brilliant Blue G250
25 % (v/v) Isopropanol
10 % (v/v) Acetic acid

6x PBJR for preparation of SDS-PAGE samples

7 ml Tris-HCl
3 ml 37 % Glycerol
0.5 M SDS
0.93 g Dithiothreitol
1.2 mg Bromophenol blue

Stacking gel for SDS-PAGE

1.3 mL rotiphorese-gel 30
6.1 mL pure water
2.5 mL 0.5 M TRIS-HCl, pH 6.8
0.1 mL 10% SDS
50 µL 10 % ammonium persulfate
5 µL TEMED

Resolving gel for SDS-PAGE

4.7 mL rotiphorese-gel 30
2.7 mL pure water
2.5 mL 0.5 M TRIS-HCl, pH 6.8
0.1 mL 10% SDS
50 µL 10 % ammonium persulfate
10 µL TEMED

SDS destaining solution

For 1 liter destaining solution:

450 mL ethanol
100 mL acetic acid
Fill the bottle with deionized H₂O

MALDI standard solution

Contains angiotensin II, substance P, adrenocorticotropic hormones, clip 1-17 and clip 18-39
Dissolve 1:25 with water.

Trypsin solution

50 µL 3 mM TRIS-HCl, pH 8.8
0.5 µL trypsin

HCCA Matrix for MALDI

711 µL 100% acetonitrile
37 µL 0.1% TFA
36 µL HCCA stock solution
8 µL 10% TFA
8 µL 100 mM NH₄H₂PO₄

PDITC solution for activation of paper

p-phenylen-diisothiocyanate
Dissolve in pure ethanol.