Team:Bielefeld-Germany/Protocols/Materials

From 2011.igem.org

Revision as of 12:22, 15 September 2011

Materials: These enzymes, kits and materials were used in our project.

Used enzymes

Used Kits

Media, buffer, solutions etc.

Ampicillin stock solution

• Solubilize 100 mg mL^-1 Ampicillin
• Store at -20 °C

Chloramphenicol stock solution

• Solubilize 20 mg mL^-1 Chloramphenicol
• Store at -20 °C

TAE buffer

For 1 L of 50 x TAE buffer you need:

• 242.48 g Tris
• 41.02 g Sodiumacetate
• 18.612 g EDTA
• Adjust pH to 7.8 with acetic acid
• Solve in dH₂O

10 mL of the stock is diluted in 1 L dH₂O for the gel electrophoresis (0.5 x TAE buffer).

DNA loading buffer

• 50 % (v/v) glycerol
• 1 mM EDTA
• 0.1 % (w/v) bromphenol blue
• Solve in ddH₂O

LB medium

For 1 L of LB medium you need:

• 10 g Trypton
• 5 g yeast extract
• 10 g NaCl
• 12 g Agar-Agar (for plates)
• Adjust pH to 7.4

Autoinduction medium for KRX

This medium is based on the LB medium.

After heat sterilization of 900 mL add the following chemicals

• 5 mL of a 200 g L^-1 steril L-rhamnose stock solution (final concentration 2 g L^-1 L-rhamnose)
• 2.5 mL of a 200 g L^-1 steril glucose stock solution (final concentration 0,5 g L^-1 glucose)
• if necessary add antibiotics
  • Cm: 1 mL of a 20 μg mL^-1 Cm stock solution (final concentration 2 μg L^-1)
  • Amp: 1 mL of a 100 μg mL^-1 Amp stock solution (final concentration 100 μg L^-1)
• fill-up to 1 L with steril ddH₂O

M9 medium

For 250 mL M9 medium you need 175 mL sterile water (for plates add 4 g Agar-Agar as well). Then add (in the following order:

• 250 µL 100 mM CaCl₂
• 2.5 mL trace salts
  • store this stock solution in the dark
• 250 µL MgSO₄
• 250 µL 50 mM FeCl₃ / 100 mM citrate (one solution, citrate is iron carrier)
  • store this stock solution cold and in the dark
• carbon source stock solution (e.g. glucose)
• 50 mL 5x M9 salts stock solution
  • 64 g L^-1 Na₂HPO₄ * 7 H₂O
  • 15 g L^-1 KH₂PO₄
  • 2.5 g L^-1 NaCl
  • 5 g L^-1 NH₄Cl
• antibiotic stock solution
• fill up to 250 mL with sterile water

HSG medium

• 14.9 g L^-1 glycerine
• 13.5 g L^-1 soy peptone
• 7 g L^-1 yeast extract
• 2.5 g L^-1 NaCl
• 2.3 g L^-1 K₂HPO₄
• 1.5 g L^-1 KH₂PO₄
• 0.249 g L^-1 MgSO₄ * 7 H₂O

5x isothermal reaction buffer for Gibson assembly

storage -20˚C

• 3 mL of 1 M Tris-HCl (pH 7.5)
• 150 µL of 2 M MgCl2,
• 60 µL of 100 mM dGTP
• 60 µL of 100 mM dATP
• 60 µL of 100 mM dTTP,
• 60 µL of 100 mM dCTP
• 300 µL of 1 M DTT
• 1.5 g PEG-8000 and
• 300 µL of 100 mM NAD

Cold osmotic shock buffers for the release of periplasmic protein fraction

Cell fractionating buffer #1 (pH 8)

• 0,2 M Tris
• 200 g L ^-1 sucrose
• 0,1 M EDTA

Cell fractionating buffer #2 (pH 8)

• 0,01 M Tris
• 0,005 M MgSO₄

Denaturation buffer for inclusion bodies

• 6 M urea
• 50 mM Tris-HCl
• 10 mM MgCl₂

Buffers for S-layer IEX

Binding Buffer:

• 25 mM Sodiumacetate, pH 6
• 25 mM NaCl

Elution Buffer:

• 25 mM Sodiumacetate, pH 6
• 1 M NaCl

SDS-PAGE gel

The following amouts are for one gel. Stacking gel 5 %:

• 775 μL H₂O
• 1.25 mL 0,25 M Tris (pH 6,8)
• 425 μL Bis/Acrylamide (0,8 %, 30 %)
• 50 μL 5 % SDS
• 25 μL 10 % Ammonium persulfate
• 3 μL TEMED

Separating gel 12 %:

• 1.5 mL H₂O
• 2.8 mL 1 M Tris (pH 8,8)
• 3.0 mL Bis/ Acrylamide (0,8%, 30%)
• 150 μL 5% SDS
• 37.5 μL 10% Ammonium persulfate
• 5 μL TEMED

SDS running buffer

• 25 mM Tris [pH 8,3]
• 192 mM Glycerol
• 0,1 % SDS

4x Laemmli-buffer

• 250 mM Tris-HCl
• 40 % [v/v] Glycerol
• 20 % [v/v] 2-Mercapthoethanol
• 80 g L^-1 SDS
• 0,04 g L^-1 BPB

Colloidal Coomassie Brilliant Blue G-250 staining solution

for 1 L staining solution

• dissolve 50 g L^-1 (NH4)₂SO₄ in ddH₂O
• add 10 % (v/v) ethanol
• dissolve 0,2 g L^-1 Coomassie Brilliant Blue G-250
• add 2 % (v/v) phosphoric acid
• fill up to 1 L with ddH₂O

Silver staining solutions

Fixation solution:

• 50 % (v/v) ethanol
• 12 % (v/v) acetic acid
• 1 mL L^-1 formaldehyde (37 %)

Thiosulfate solution:

• 0.1 g L^-1 Na₂S₂O₃

Impregnation solution:

• 2 g L^-1 silver nitrate (AgNO₃)
• 0.75 mL L^-1 formaldehyde (37 %)

Developing solution:

• 120 g L^-1 sodium carbonate (Na₂CO₃)
• 1 mL L^-1 formaldehyde (37 %)

Stop solution:

• 18.6 g L^-1 EDTA

Binding buffer

• 20 mM Na₃PO₄, pH 7,4
• 500 mM NaCl
• 20 mM Imidazole

NPI-10 (lysis buffer)

• 50 mM NaH₂PO₄, pH 8,0
• 300 mM NaCl
• 10 mM Imidazole
• 2 mM PMSF

NPI-20 (wash buffer)

• 50 mM NaH₂PO₄, pH 8,0
• 300 mM NaCl
• 20 mM Imidazole

NPI-500 (elution buffer)

• 50 mM NaH₂PO₄, pH 8,0
• 300 mM NaCl
• 500 mM Imidazole

Deadenylation buffer

• 20 mM Tris-HCl, pH 7,5
• 50 mM NaCl
• 4 mM MgCl₂
• 1 mM EDTA
• 1 mM DTT
• 1 mM NMN

DNA ligase buffer

• 20 mM Tris-HCl, pH 7,5
• 50 mM NaCl
• 20 % [v/v] Glycerol

NAD⁺ bioassay buffer

• 50 mM Tris-HCl, pH 8,0
• 10 mM MgCl₂
• 2,5 mM CaCl₂
• 5 mM DTT
• 0.05 % BSA

Buffers for His-Tag affinity chromatography

• For denaturing conditions add 6 M Urea
• Adjust pH to 7,4 - 7,6

Used chemicals