Team:Bielefeld-Germany/Protocols/Materials

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Revision as of 14:07, 9 September 2011

Contents

Used enzymes

Enzyme Producer
AgeI [http://www.fermentas.de/product_info.php?info=p247 Fermentas]
DpnI [http://www.fermentas.de/product_info.php?info=p296 Fermentas]
EcoRI [http://www.fermentas.de/product_info.php?info=p336 Fermentas]
GoTaq DNA-polymerase [http://www.promega.com/products/pcr/routine-pcr/gotaq-pcr-core-systems/ Promega]
KOD Hotstart DNA-polymerase [http://www.merck-chemicals.com/germany/life-science-research/kod-hot-start-dna-polymerase/EMD_BIO-71086/p_iFCb.s1O874AAAEj2Bl9.zLX Novagen]
NgoMIV [http://www.neb.com/nebecomm/products/productR0564.asp NEB]
OneTaq DNA-polymerase [http://www.neb.com/nebecomm/products/productM0480.asp NEB]
Pfu DNA-polymerase [http://www.promega.com/products/pcr/routine-pcr/pfu-dna-polymerase/ Promega]
PstI [http://www.fermentas.de/product_info.php?info=p458 Fermentas]
Phusion HF DNA-polymerase [http://www.finnzymes.com/pcr/phusion_high_fidelity_dna_polymerase.html Finnzymes]
Shrimp alcaline phosphatase [http://www.fermentas.de/product_info.php?info=p592 Fermentas]
SpeI [http://www.fermentas.de/product_info.php?info=p202 Fermentas]
T4-DNA-Ligase [http://www.fermentas.de/product_info.php?info=p580 Fermentas]
T5 exonuclease [http://www.neb.com/nebecomm/products/productM0363.asp NEB]
taq DNA Ligase [http://www.neb.com/nebecomm/products/productM0208.asp NEB]
taq DNA-polymerase [http://www.bioline.com/h_prod_detail.asp?itemid=219 Bioline]


Used Kits

Function Name
Molecular Cloning [http://www.fermentas.de/product_info.php?info=p1620 Fermentas CloneJET™ PCR Cloning Kit]
Plasmid purification [http://www.fermentas.de/product_info.php?info=p874 Fermentas GeneJET™ Plasmid Miniprep Kit]
Plasmid purification [http://www.promega.com/b/de/minis/default.aspx?utm_source=Promega&utm_medium=Online&utm_campaign=Online_DEPureYield100Preps_Promega_HP_Banner Promega PureYield™ Plasmid Preps]
PCR Cleanup [http://www.mn-net.com/tabid/10745/default.aspx Macherey Nagel NucleoSpin® Extract II]
PCR Cleanup [http://www.promega.com/products/dna-and-rna-purification/dna-fragment-purification/wizard-sv-gel-and-pcr-clean_up-system/ Promega Wizard® SV Gel and PCR Clean-Up]
PCR core system [http://www.promega.com/products/pcr/routine-pcr/gotaq-pcr-core-systems/ 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


SDS-PAGE gel

The amouts in following tabe are for one gel.

Type of gel Component Volume
stacking gel 5 % H2O 775 μL
0,25 M Tris (pH 6,8) 1,25 mL
Bis/Acrylamide (0,8 %, 30 %) 425 μL
5 % SDS 50 μL
10 % Ammonium persulfate 25 μL
TEMED 3 μL
separating gel 12 % H2O 1,5 mL
1 M Tris (pH 8,8) 2,8 mL
Bis/ Acrylamide (0,8%, 30%) 3,0 mL
5% SDS 150 μL
10% Ammonium persulfate 37,5 μL
TEMED 5 μL


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


Used chemicals

Chemical Producer Purity
Acetonitrile VWR 99.9 %, HPLC Grade
Bisphenol A [http://www.sigmaaldrich.com/catalog/Lookup.do?N5=All&N3=mode+matchpartialmax&N4=bisphenol+a&D7=0&D10=bisphenol+a&N1=S_ID&ST=RS&N25=0&F=PR Sigma] 97 %
Bisphenol F [http://www.alfa.com/de/GP100W.pgm?DSSTK=A11417&rnd=097380668 Alfa Aesar] 98 %
Ethylacetate VWR > 99.5 %, p.a.
Chemical A [http://www.link.com Producer A] XX.X %
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