Team:Bielefeld-Germany/Protocols/Materials

From 2011.igem.org

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(Deadenylation buffer)
 
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{{Bielefeld_2011_Header}}
{{Bielefeld_2011_Header}}
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<html><img src="http://2011.igem.org/wiki/images/7/72/Bielefeld-header-protocols-materials.png"/><p></p></html>
'''Materials:''' These enzymes, kits and materials were used in our project.
'''Materials:''' These enzymes, kits and materials were used in our project.
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| ''taq'' DNA-polymerase ||  style="padding-left:5px;" |[http://www.bioline.com/h_prod_detail.asp?itemid=219 Bioline]
| ''taq'' DNA-polymerase ||  style="padding-left:5px;" |[http://www.bioline.com/h_prod_detail.asp?itemid=219 Bioline]
|-
|-
 +
| XbaI ||  style="padding-left:5px;" |[http://www.fermentas.de/product_info.php?info=p247 Fermentas]
|}
|}
-
 
==Used Kits==
==Used Kits==
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===Chloramphenicol stock solution===
===Chloramphenicol stock solution===
-
* Solubilize 20 mg mL<sup>-1</sup> Chloramphenicol
+
* Solubilize 20 mg mL<sup>-1</sup> Chloramphenicol in 100 % Ethanol
* Store at -20 °C
* Store at -20 °C
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*0.01 M Tris
*0.01 M Tris
*0.005 M MgSO<sub>4</sub>
*0.005 M MgSO<sub>4</sub>
 +
===Denaturation buffer for inclusion bodies===
===Denaturation buffer for inclusion bodies===
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===Buffers for S-layer IEX===
===Buffers for S-layer IEX===
-
Binding Buffer:  
+
Binding Buffer ''Corynebacterium'' and ''L. sphaericus'':  
* 25 mM Sodiumacetate, pH 6
* 25 mM Sodiumacetate, pH 6
* 25 mM NaCl
* 25 mM NaCl
-
Elution Buffer:
+
Binding Buffer ''G. stearothermophilus'':
 +
* 25 mM Sodiumphosphate, pH 7
 +
* 25 mM NaCl
 +
 
 +
Elution Buffer ''Corynebacterium'' and ''L. sphaericus'':
* 25 mM Sodiumacetate, pH 6
* 25 mM Sodiumacetate, pH 6
* 1 M NaCl
* 1 M NaCl
 +
 +
Elution Buffer ''G. stearothermophilus'':
 +
* 25 mM Sodiumphosphate, pH 7
 +
* 1 M NaCl
 +
 +
 +
===Buffers for S-layer HIC===
 +
Binding Buffer:
 +
* 50 mM Ammoniumphosphate, pH 7.0
 +
* 1200 mM Ammoniumsulfate
 +
 +
Elution Buffer:
 +
* 50 mM Ammoniumphosphate, pH 7.0
 +
 +
 +
===Hanks Buffered Saline Solution (HBSS)===
 +
 +
*0.137 M NaCl
 +
*5.4 mM KCl
 +
*0.25 mM Na<sub>2</sub>HPO<sub>4</sub>
 +
*0.44 mM KH<sub>2</sub>PO<sub>4</sub>
 +
*1.3 mM CaCl<sub>2</sub>
 +
*1.0 mM MgSO<sub>4</sub>
 +
*4.2 mM NaHCO<sub>3</sub>
 +
 +
 +
===Recrystallization buffer SbpA===
 +
 +
* 0.5 mM Tris-HCl, pH 9.0
 +
* 10 mM CaCl<sub>2</sub>
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*25 μL 10 % Ammonium persulfate
*25 μL 10 % Ammonium persulfate
*3 μL TEMED
*3 μL TEMED
-
 
Separating gel 12 %:
Separating gel 12 %:
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*192 mM Glycerol
*192 mM Glycerol
*0.1 % SDS
*0.1 % SDS
 +
===4x Laemmli-buffer===
===4x Laemmli-buffer===
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*20 % [v/v] 2-Mercapthoethanol
*20 % [v/v] 2-Mercapthoethanol
*80 g L<sup>-1</sup> SDS
*80 g L<sup>-1</sup> SDS
-
*0,04 g L<sup>-1</sup> BPB
+
*0.04 g L<sup>-1</sup> BPB
 +
 
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*dissolve 50 g L<sup>-1</sup> (NH4)<sub>2</sub>SO<sub>4</sub> in ddH<sub>2</sub>O
*dissolve 50 g L<sup>-1</sup> (NH4)<sub>2</sub>SO<sub>4</sub> in ddH<sub>2</sub>O
*add 10 % (v/v) ethanol
*add 10 % (v/v) ethanol
-
*dissolve 0,2 g L<sup>-1</sup> Coomassie Brilliant Blue G-250
+
*dissolve 0.2 g L<sup>-1</sup> Coomassie Brilliant Blue G-250
*add 2 % (v/v) phosphoric acid
*add 2 % (v/v) phosphoric acid
*fill up to 1 L with ddH<sub>2</sub>O
*fill up to 1 L with ddH<sub>2</sub>O
 +
 +
 +
=== Fairbanks Coomassie staining solutions===
 +
Solution A:
 +
*25 % (v/v) ispropanol
 +
*10 % (v/v) acetic acid
 +
*0,5 g L<sup>-1</sup> Coomassie Brilliant Blue R-250
 +
 +
Solution B:
 +
*10 % (v/v) ispropanol
 +
*10 % (v/v) acetic acid
 +
*0,05 g L<sup>-1</sup> Coomassie Brilliant Blue R-250
 +
 +
Solution C:
 +
*10 % (v/v) acetic acid
 +
*0,025 g L<sup>-1</sup> Coomassie Brilliant Blue R-250
 +
 +
Solution D:
 +
*10 % (v/v) acetic acid
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*12 % (v/v) acetic acid
*12 % (v/v) acetic acid
*1 mL L<sup>-1</sup> formaldehyde (37 %)
*1 mL L<sup>-1</sup> formaldehyde (37 %)
-
 
Thiosulfate solution:
Thiosulfate solution:
*0.1 g L<sup>-1</sup> Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub>
*0.1 g L<sup>-1</sup> Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub>
-
 
Impregnation solution:
Impregnation solution:
*2 g L<sup>-1</sup> silver nitrate (AgNO<sub>3</sub>)
*2 g L<sup>-1</sup> silver nitrate (AgNO<sub>3</sub>)
*0.75 mL L<sup>-1</sup> formaldehyde (37 %)
*0.75 mL L<sup>-1</sup> formaldehyde (37 %)
-
 
Developing solution:
Developing solution:
*120 g L<sup>-1</sup> sodium carbonate (Na<sub>2</sub>CO<sub>3</sub>)
*120 g L<sup>-1</sup> sodium carbonate (Na<sub>2</sub>CO<sub>3</sub>)
*1 mL L<sup>-1</sup> formaldehyde (37 %)
*1 mL L<sup>-1</sup> formaldehyde (37 %)
-
 
Stop solution:
Stop solution:
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-
===Binding buffer===
+
===Enzyme buffer===
-
* 20 mM Na<sub>3</sub>PO<sub>4</sub>, pH 7,4
+
*10 mM MgCl<sub>2</sub>
-
* 500 mM NaCl
+
*50 mM Tris/HCl
-
* 20 mM Imidazole
+
*pH 7.5
===NPI-10 (lysis buffer)===
===NPI-10 (lysis buffer)===
-
* 50 mM NaH<sub>2</sub>PO<sub>4</sub>, pH 8,0
+
* 50 mM NaH<sub>2</sub>PO<sub>4</sub>, pH 8.0
* 300 mM NaCl
* 300 mM NaCl
* 10 mM Imidazole
* 10 mM Imidazole
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===NPI-20 (wash buffer)===
===NPI-20 (wash buffer)===
-
* 50 mM NaH<sub>2</sub>PO<sub>4</sub>, pH 8,0
+
* 50 mM NaH<sub>2</sub>PO<sub>4</sub>, pH 8.0
* 300 mM NaCl
* 300 mM NaCl
* 20 mM Imidazole
* 20 mM Imidazole
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===NPI-500 (elution buffer)===
===NPI-500 (elution buffer)===
-
* 50 mM NaH<sub>2</sub>PO<sub>4</sub>, pH 8,0
+
* 50 mM NaH<sub>2</sub>PO<sub>4</sub>, pH 8.0
* 300 mM NaCl
* 300 mM NaCl
* 500 mM Imidazole
* 500 mM Imidazole
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* 1 mM EDTA
* 1 mM EDTA
* 1 mM DTT
* 1 mM DTT
-
* 1 mM NMN
+
* 10 mM NMN
 +
 
===DNA ligase buffer===
===DNA ligase buffer===
-
* 20 mM Tris-HCl, pH 7,5
+
* 20 mM Tris-HCl, pH 7.5
* 50 mM NaCl
* 50 mM NaCl
* 20 % [v/v] Glycerol
* 20 % [v/v] Glycerol
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===NAD<sup>+</sup> bioassay buffer===
===NAD<sup>+</sup> bioassay buffer===
-
* 50 mM Tris-HCl, pH 8,0
+
* 50 mM Tris-HCl, pH 8.0
* 10 mM MgCl<sub>2</sub>
* 10 mM MgCl<sub>2</sub>
* 2.5 mM CaCl<sub>2</sub>
* 2.5 mM CaCl<sub>2</sub>
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*For denaturing conditions add 6 M Urea
*For denaturing conditions add 6 M Urea
*Adjust pH to 7.4 - 7.6
*Adjust pH to 7.4 - 7.6
-
{| border="1"
+
{| class="wikitable" style="text-align:left"
! Buffer
! Buffer
! Sodium phosphate [mM]
! Sodium phosphate [mM]
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|-
|-
|}
|}
-
 
-
<br style="clear: both" />
 
==Used chemicals==
==Used chemicals==
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| L-rhamnose || style="padding-left:5px;" | [http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=de&N4=83650|FLUKA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC Fluka] || style="padding-left:5px;" | ≥ 99 %
| L-rhamnose || style="padding-left:5px;" | [http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=de&N4=83650|FLUKA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC Fluka] || style="padding-left:5px;" | ≥ 99 %
|-
|-
-
| Chemical A || style="padding-left:5px;" | [http://www.link.com Producer A] || style="padding-left:5px;" | XX.X %
+
| Trypsin || style="padding-left:5px;" | [http://www.promega.com/products/protein-expression-and-analysis/mass-spectrometry-analysis/sequencing-grade-modified-trypsin/ Promega] || style="padding-left:5px;" | n/a
-
|-
+
|}
|}

Latest revision as of 17:28, 25 October 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
XbaI Fermentas

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 in 100 % Ethanol
  • 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 Corynebacterium and L. sphaericus:

  • 25 mM Sodiumacetate, pH 6
  • 25 mM NaCl

Binding Buffer G. stearothermophilus:

  • 25 mM Sodiumphosphate, pH 7
  • 25 mM NaCl

Elution Buffer Corynebacterium and L. sphaericus:

  • 25 mM Sodiumacetate, pH 6
  • 1 M NaCl

Elution Buffer G. stearothermophilus:

  • 25 mM Sodiumphosphate, pH 7
  • 1 M NaCl


Buffers for S-layer HIC

Binding Buffer:

  • 50 mM Ammoniumphosphate, pH 7.0
  • 1200 mM Ammoniumsulfate

Elution Buffer:

  • 50 mM Ammoniumphosphate, pH 7.0


Hanks Buffered Saline Solution (HBSS)

  • 0.137 M NaCl
  • 5.4 mM KCl
  • 0.25 mM Na2HPO4
  • 0.44 mM KH2PO4
  • 1.3 mM CaCl2
  • 1.0 mM MgSO4
  • 4.2 mM NaHCO3


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


Fairbanks Coomassie staining solutions

Solution A:

  • 25 % (v/v) ispropanol
  • 10 % (v/v) acetic acid
  • 0,5 g L-1 Coomassie Brilliant Blue R-250

Solution B:

  • 10 % (v/v) ispropanol
  • 10 % (v/v) acetic acid
  • 0,05 g L-1 Coomassie Brilliant Blue R-250

Solution C:

  • 10 % (v/v) acetic acid
  • 0,025 g L-1 Coomassie Brilliant Blue R-250

Solution D:

  • 10 % (v/v) acetic acid


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


Enzyme buffer

  • 10 mM MgCl2
  • 50 mM Tris/HCl
  • pH 7.5


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 %
Trypsin Promega n/a