Team:Bielefeld-Germany/Protocols/Materials
From 2011.igem.org
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{{Bielefeld_2011_Header}} | {{Bielefeld_2011_Header}} | ||
+ | <html><img src="https://static.igem.org/mediawiki/2011/7/72/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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After heat sterilization of 900 mL add the following chemicals | After heat sterilization of 900 mL add the following chemicals | ||
* 5 mL of a 200 g L<sup>-1</sup> steril L-rhamnose stock solution (final concentration 2 g L<sup>-1</sup> L-rhamnose) | * 5 mL of a 200 g L<sup>-1</sup> steril L-rhamnose stock solution (final concentration 2 g L<sup>-1</sup> L-rhamnose) | ||
- | * 2.5 mL of a 200 g L<sup>-1</sup> steril glucose stock solution (final concentration 0 | + | * 2.5 mL of a 200 g L<sup>-1</sup> steril glucose stock solution (final concentration 0.5 g L<sup>-1</sup> glucose) |
* if necessary add antibiotics | * if necessary add antibiotics | ||
** Cm: 1 mL of a 20 μg mL<sup>-1</sup> [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#Chloramphenicol_stock_solution Cm stock solution] (final concentration 2 μg L<sup>-1</sup>) | ** Cm: 1 mL of a 20 μg mL<sup>-1</sup> [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#Chloramphenicol_stock_solution Cm stock solution] (final concentration 2 μg L<sup>-1</sup>) | ||
** Amp: 1 mL of a 100 μg mL<sup>-1</sup> [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#Ampicillin_stock_solution Amp stock solution] (final concentration 100 μg L<sup>-1</sup>) | ** Amp: 1 mL of a 100 μg mL<sup>-1</sup> [https://2011.igem.org/Team:Bielefeld-Germany/Protocols#Ampicillin_stock_solution Amp stock solution] (final concentration 100 μg L<sup>-1</sup>) | ||
* fill-up to 1 L with steril ddH<sub>2</sub>O | * fill-up to 1 L with steril ddH<sub>2</sub>O | ||
+ | |||
===M9 medium=== | ===M9 medium=== | ||
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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 | + | *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 | + | *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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- | === | + | ===Enzyme buffer=== |
- | * | + | *10 mM MgCl<sub>2</sub> |
- | * | + | *50 mM Tris/HCl |
- | * | + | *pH 7.5 |
===NPI-10 (lysis buffer)=== | ===NPI-10 (lysis buffer)=== | ||
- | * 50 mM NaH<sub>2</sub>PO<sub>4</sub>, pH 8 | + | * 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 | + | * 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 | + | * 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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===Deadenylation buffer=== | ===Deadenylation buffer=== | ||
- | * 20 mM Tris-HCl, pH 7 | + | * 20 mM Tris-HCl, pH 7.5 |
* 50 mM NaCl | * 50 mM NaCl | ||
* 4 mM MgCl<sub>2</sub> | * 4 mM MgCl<sub>2</sub> | ||
* 1 mM EDTA | * 1 mM EDTA | ||
* 1 mM DTT | * 1 mM DTT | ||
- | * | + | * 10 mM NMN |
===DNA ligase buffer=== | ===DNA ligase buffer=== | ||
- | * 20 mM Tris-HCl, pH 7 | + | * 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 | + | * 50 mM Tris-HCl, pH 8.0 |
* 10 mM MgCl<sub>2</sub> | * 10 mM MgCl<sub>2</sub> | ||
- | * 2 | + | * 2.5 mM CaCl<sub>2</sub> |
* 5 mM DTT | * 5 mM DTT | ||
* 0.05 % BSA | * 0.05 % BSA | ||
+ | |||
===Buffers for His-Tag affinity chromatography=== | ===Buffers for His-Tag affinity chromatography=== | ||
*For denaturing conditions add 6 M Urea | *For denaturing conditions add 6 M Urea | ||
- | *Adjust pH to 7 | + | *Adjust pH to 7.4 - 7.6 |
- | {| | + | {| class="wikitable" style="text-align:left" |
! Buffer | ! Buffer | ||
! Sodium phosphate [mM] | ! Sodium phosphate [mM] | ||
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|- | |- | ||
|} | |} | ||
- | |||
- | |||
==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 % | ||
|- | |- | ||
- | | | + | | 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
![](https://static.igem.org/mediawiki/2011/3/3c/Bielefeld-Germany2011-header-full.png)
![](https://static.igem.org/mediawiki/2011/7/72/Bielefeld-header-protocols-materials.png)
Materials: These enzymes, kits and materials were used in our project.
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
- 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 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 |