Team:Bielefeld-Germany/Protocols/Materials

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

Revision as of 13:08, 20 September 2011

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

Contents

Used enzymes

Enzyme Producer
AgeI Fermentas
DpnI Fermentas
EcoRI Fermentas
GoTaq DNA-polymerase Promega
KOD Hotstart DNA-polymerase Novagen
NgoMIV NEB
OneTaq DNA-polymerase NEB
Pfu DNA-polymerase Promega
PstI Fermentas
Phusion HF DNA-polymerase Finnzymes
Shrimp alcaline phosphatase Fermentas
SpeI Fermentas
T4-DNA-Ligase Fermentas
T5 exonuclease NEB
taq DNA Ligase NEB
taq DNA-polymerase Bioline


Used Kits

Function Name
Molecular Cloning Fermentas CloneJET™ PCR Cloning Kit
Plasmid purification Fermentas GeneJET™ Plasmid Miniprep Kit
Plasmid purification Promega PureYield™ Plasmid Preps
PCR Cleanup Macherey Nagel NucleoSpin® Extract II
PCR Cleanup Promega Wizard® SV Gel and PCR Clean-Up
PCR core system Promega GoTaq® PCR Core System I

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
  • 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


Hanks Buffered Saline Solution (HBSS)

Receipe...


Recrystallization buffer SbpA

  • 0.5 mM Tris-HCl, pH 9.0
  • 10 mM CaCl2


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
  • 10 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
Buffer Sodium phosphate [mM] NaCl [mM] Imidazole [mM]
Binding buffer 20 500 5
Elution buffer 1 20 500 40
Elution buffer 2 20 500 60
Elution buffer 3 20 500 100
Elution buffer 4 20 500 300
Elution buffer 5 20 500 500


Used chemicals

Chemical Producer Purity
Acetonitrile VWR 99.9 %, HPLC Grade
Bisphenol A Sigma 97 %
Bisphenol F Alfa Aesar 98 %
Ethylacetate VWR > 99.5 %, p.a.
Isopropyl β-D-1-thiogalactopyranoside Roth ≥ 99 %
L-rhamnose Fluka ≥ 99 %
Chemical A Producer A XX.X %