Team:Heidelberg LSL/Notebook materials
From 2012hs.igem.org
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+ | <h4>Kits for purification of plasmid-DNA and DNA fragments</h4> | ||
+ | |||
+ | <p>DNA was extracted in small or medium scale using the Promega or Qiagen DNA preparation kits. For all DNA extractions before digestions/transformations, Promega Kits were applied. Only when performing sequencing, Qiagen kits were preferred.</p> | ||
- | |||
<center> | <center> | ||
- | <table class="wikitable sortable" border="0" style="text-align:left > | + | <table class="wikitable sortable" border="0" style="text-align:left" width="600px"> |
<caption align="top, left"> | <caption align="top, left"> | ||
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<a href="http://www.promega.com/b/de/aufreinigung/minis/default.aspx">PureYield™ Plasmid Miniprep System | <a href="http://www.promega.com/b/de/aufreinigung/minis/default.aspx">PureYield™ Plasmid Miniprep System | ||
</a> | </a> | ||
- | </p></td><td>small scale plasmid purification from E. coli Top10 cells before digestion or transformation into different E. coli strains </td><td>Promega</td> | + | </p></td><td>small scale plasmid purification from <i>E. coli</i> Top10 cells before digestion or transformation into different <i>E. coli</i> strains </td><td>Promega</td> |
</tr> | </tr> | ||
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<a href="http://www.qiagen.com/dm/aw/miniprep/default.aspx?gaw=QVenGA3Miniprep&gkw=miniprep+kit">QIAprep Spin MiniPrep Kit (50) | <a href="http://www.qiagen.com/dm/aw/miniprep/default.aspx?gaw=QVenGA3Miniprep&gkw=miniprep+kit">QIAprep Spin MiniPrep Kit (50) | ||
</a> | </a> | ||
- | </p></td><td>small scale plasmid purification from E. coli Top10 cells before sequencing </td><td>Qiagen</td> | + | </p></td><td>small scale plasmid purification from <i>E. coli</i> Top10 cells before sequencing </td><td>Qiagen</td> |
</tr> | </tr> | ||
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<a href="http://www.promega.com/products/dna-and-rna-purification/plasmid-purification/pureyield-plasmid-midiprep-system/">PureYield™ Plasmid Midiprep System | <a href="http://www.promega.com/products/dna-and-rna-purification/plasmid-purification/pureyield-plasmid-midiprep-system/">PureYield™ Plasmid Midiprep System | ||
</a> | </a> | ||
- | </p></td><td>medium scale plasmid purification from E. coli Top10 cells before digestion or transformation into different E. coli strains </td><td>Promega</td> | + | </p></td><td>medium scale plasmid purification from <i>E. coli</i> Top10 cells before digestion or transformation into different <i>E. coli</i> strains </td><td>Promega</td> |
</tr> | </tr> | ||
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</center> | </center> | ||
+ | <h4>LB growth media - components an recipe</h4> | ||
+ | |||
+ | <p>LB media was prepared from 10 g/l tryptone, 5 g/l yeast extract and 5 g/l NaCl and autoclaved before usage. For preparing LB agar plates, 1.5 g of agar were added to the media. The media was furthermore substituted with 100 ug/ml ampicillin (for LB-amp) or 35 ug/ul chloramphenicol (for LB-cm).</p> | ||
+ | |||
+ | |||
+ | <center> | ||
+ | <table class="wikitable sortable" border="0" style="text-align:left" width="600px" > | ||
+ | |||
+ | <caption align="top, left"> | ||
+ | </caption><tr bgcolor="#cccccc"> | ||
+ | <td> Name</td><td>Usage</td><td>Company</td></tr> | ||
+ | |||
+ | <tr> | ||
+ | <td><p> | ||
+ | <a href="http://www.bd.com/ds/productCenter/211701.asp">Trypton | ||
+ | </a> | ||
+ | </p></td><td>LB media </td><td>BD</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td><p> | ||
+ | <a href="http://www.bd.com/ds/productCenter/288620.asp">Yeast Extract | ||
+ | </a> | ||
+ | </p></td><td>LB media </td><td>BD</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td><p> | ||
+ | <a href="http://www.sigmaaldrich.com/programs/research-essentials-products.html?TablePage=102880799">NaCl | ||
+ | </a> | ||
+ | </p></td><td>LB media </td><td>Sigma</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td><p> | ||
+ | <a href="http://www.sigmaaldrich.com/analytical-chromatography/microbiology/basic-ingredients/agar.html">Agar | ||
+ | </a> | ||
+ | </p></td><td>preparing agar plates </td><td>Sigma</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td><p> | ||
+ | <a href="http://www.sigmaaldrich.com/catalog/search?interface=All&term=ampicillin&lang=de®ion=DE&focus=product&N=0+220003048+219853101+219853286&mode=match%20partialmax">Ampicillin | ||
+ | </a> | ||
+ | </p></td><td>usage: 100 ug/ml, selection of plasmid containing an amp resistance gene </td><td>Sigma</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td><p> | ||
+ | <a href="http://www.sigmaaldrich.com/catalog/product/sigma/c3175?lang=de®ion=DE">Chloramphenicol | ||
+ | </a> | ||
+ | </p></td><td>usage: 35 ug/ml, selection of plasmid containing an Cm resistance gene </td><td>Sigma</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td><p> | ||
+ | <a href="http://www.sigmaaldrich.com/labware/labware-products.html?TablePage=17951483">petri dish (10 cm) | ||
+ | </a> | ||
+ | </p></td><td>preparing agar plates </td><td>Sigma</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td><p> | ||
+ | <a href="http://www.sigmaaldrich.com/catalog/product/sial/b4252?lang=de®ion=DE">X-Gal | ||
+ | </a> | ||
+ | </p></td><td>dissolve in DMSO at 20 mg/ml. final concentration for blue-white screening: 100 ug/ml and for X-Gal assays in liquid LB culture: 200 ug/ml</td><td>Sigma</td> | ||
+ | </tr> | ||
+ | |||
+ | |||
+ | </table> | ||
+ | </center> | ||
+ | |||
+ | <br> | ||
+ | |||
+ | <br> | ||
+ | |||
+ | <h4><i>E. coli</i> Strains</h4> | ||
+ | |||
+ | <p><i>E. coli</i> Top10 were used for amplification of plasmid DNA and subsequent DNA substraction. Bl21(DE3) were used in all measurments/assays performed.</p> | ||
+ | |||
+ | |||
+ | <center> | ||
+ | <table class="wikitable sortable" border="0" style="text-align:left" width="600px" > | ||
+ | |||
+ | <caption align="top, left"> | ||
+ | </caption><tr bgcolor="#cccccc"> | ||
+ | <td> Strain</td><td>Genotype</td></tr> | ||
+ | |||
+ | <tr> | ||
+ | <td><p> | ||
+ | <a href="http://openwetware.org/wiki/E._coli_genotypes">Top10 | ||
+ | </a> | ||
+ | </p></td><td>F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(StrR) endA1 λ-</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td><p> | ||
+ | <a href="http://openwetware.org/wiki/E._coli_genotypes">Bl21(DE3) | ||
+ | </a> | ||
+ | </p></td><td>F– ompT gal dcm lon hsdSB(rB- mB-) λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) </td> | ||
+ | </tr> | ||
+ | |||
+ | |||
+ | </table> | ||
+ | </center> | ||
+ | |||
+ | <br> | ||
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<center> | <center> | ||
- | <table class="wikitable sortable" border="0" style="text-align:left > | + | <table class="wikitable sortable" border="0" style="text-align:left" width="600px" > |
<caption align="top, left"> | <caption align="top, left"> | ||
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<tr> | <tr> | ||
<td><p> | <td><p> | ||
- | <a href="http://www.promega.com/products/cloning-and-dna-markers/restriction-enzymes/xbai/"> | + | <a href="http://www.promega.com/products/cloning-and-dna-markers/restriction-enzymes/xbai/">XbaI |
</a> | </a> | ||
</p></td><td>restriction enzyme, recognition sequence: TCTAGA</td><td>Promega or NEB</td> | </p></td><td>restriction enzyme, recognition sequence: TCTAGA</td><td>Promega or NEB</td> | ||
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<a href="http://www.promega.com/products/cloning-and-dna-markers/restriction-enzymes/psti/">PstI | <a href="http://www.promega.com/products/cloning-and-dna-markers/restriction-enzymes/psti/">PstI | ||
</a> | </a> | ||
- | </p></td><td>restriction | + | </p></td><td>restriction enzyme, recognition sequence: CTGCAG</td><td>Promega or NEB</td> |
</tr> | </tr> | ||
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</center> | </center> | ||
+ | <br> | ||
<h4>Oligonucleotides</h4> | <h4>Oligonucleotides</h4> | ||
<p>Oligos were annealed and subsequently cloned into EcoRI/XbaI predigested reporter backbones. Thereby short synthetic DNA-sequences (in this case for the promoters psulA, precA and precB) can be generated in a fast and easy manner.</p> | <p>Oligos were annealed and subsequently cloned into EcoRI/XbaI predigested reporter backbones. Thereby short synthetic DNA-sequences (in this case for the promoters psulA, precA and precB) can be generated in a fast and easy manner.</p> | ||
- | + | ||
<center> | <center> | ||
- | <table class="wikitable sortable" border="0" style="text-align:left > | + | <table class="wikitable sortable" border="0" style="text-align:left" width="600px" > |
<caption align="top, left"> | <caption align="top, left"> | ||
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<td> Oligo Name</td><td>Oligo Sequence</td><td>sequence length</td></tr> | <td> Oligo Name</td><td>Oligo Sequence</td><td>sequence length</td></tr> | ||
+ | <tr> | ||
+ | <p> | ||
+ | <td>psulA_fw</td><td>5'-aattcgcggccgcttctagagGGGTTGATCTTTGTTGT CACTGGATGTACTGTACATCCATACAGTAACTCACc-3'</td><td>74</td> | ||
+ | </p> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <p> | ||
+ | <td>psulA_rev</td><td>5'-ctaggGTGAGTTACTGTATGGATGTACAGTACATCCAG TGACAACAAAGATCAACCCctctagaagcggccgcg-3'</td><td>74</td> | ||
+ | </p> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <p> | ||
+ | <td>precB_fw</td><td>5'aattcgcggccgcttctagagCCTGAAGGCTGGAAAGTGTGGGAGA ACGTCAGCGCGTTGCAGCAAACAATGCCCCTGATGAGTGAAAAGAc-3'</td><td>92</td> | ||
+ | </p> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <p> | ||
+ | <td>precB_rev</td><td>5'-ctaggTCTTTTCACTCATCAGGGGCATTGTTTGCTGCAACGCGCTG ACGTTCTCCCACACTTTCCAGCCTTCAGGctctagaagcggccgcg-3'</td><td>92</td> | ||
+ | </p> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <p> | ||
+ | <td>precC_fw</td><td>5'-aattcgcggccgcttctagagTTCACCCGGGGGCAGAGAAGGC GAGATGACCCGCCTGCATTGCCCGAATCGTCAGTAGTCAGGAGCCGCTc-3'</td><td>92</td> | ||
+ | </p> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <p> | ||
+ | <td>precC_rev</td><td>5'-ctaggAGCGGCTCCTGACTACTGACGATTCGGGCAATGCAGGCG GGTCATCTCGCCTTCTCTGCCCCCGGGTGAActctagaagcggccgcg-3'</td><td>92</td> | ||
+ | </p> | ||
+ | </tr> | ||
+ | |||
+ | </table> | ||
+ | </center> | ||
+ | |||
+ | <br> | ||
+ | |||
+ | |||
+ | |||
+ | <h4>Gel electrophoresis</h4> | ||
+ | |||
+ | <p>For gel electrophoresis we used 1 % agarose dissolved in 1x TAE buffer. Gels were run at 110 V for 25-60 min.</p> | ||
+ | |||
+ | |||
+ | <center> | ||
+ | <table class="wikitable sortable" border="0" style="text-align:left" width="600px" > | ||
+ | |||
+ | <caption align="top, left"> | ||
+ | </caption><tr bgcolor="#cccccc"> | ||
+ | <td> Name</td><td>Usage</td><td>Company</td></tr> | ||
+ | |||
+ | <tr> | ||
+ | <td><p> | ||
+ | <a href="http://www.sigmaaldrich.com/catalog/search?interface=All&term=agarose&lang=de®ion=DE&focus=product&N=0+220003048+219853101+219853286&mode=match%20partialmax">Agarose | ||
+ | </a> | ||
+ | </p></td><td>to be dissolved to 1 % in 1x TAE buffer </td><td>Sigma</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td><p> | ||
+ | <a href="http://www.sigmaaldrich.com/catalog/search?interface=All&term=TAE+buffer&lang=de®ion=DE&focus=product&N=0+220003048+219853101+219853286&mode=match%20partialmax">TAE buffer | ||
+ | </a> | ||
+ | </p></td><td>Tris-Acetate-EDTA buffer for gel electrophoresis </td><td>Sigma</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td><p> | ||
+ | <a href="http://www.promega.com/products/biochemicals-and-labware/biochemical-buffers-and-reagents/blue_orange-loading-dye_-6x/">Blue/Orange loading dye, 6x | ||
+ | </a> | ||
+ | </p></td><td>dissolve to 1x in DNA samples before loading onto agarose gel </td><td>Promega</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td><p> | ||
+ | <a href="http://www.promega.com/products/cloning-and-dna-markers/molecular-weight-markers/dna-ladders/">1kb DNA ladder | ||
+ | </a> | ||
+ | </p></td><td>1kb DNA ladder </td><td>Promega</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td><p> | ||
+ | <a href="http://www.fermentas.com/en/products/all/dna-electrophoresis/generuler-dna-ladders/sm033-generuler-mix">GeneRuler DNA ladder Mix | ||
+ | </a> | ||
+ | </p></td><td>DNA ladder mix, 100 bp - 10 kb </td><td>Fermentas</td> | ||
+ | </tr> | ||
+ | |||
+ | |||
+ | </table> | ||
+ | </center> | ||
+ | |||
+ | <br> | ||
+ | |||
+ | |||
+ | <h4>Measurements</h4> | ||
+ | |||
+ | <p>The following materials and devices were used for our measurements and assays (DNA concentration, ONPG- and X-Gal assay) </p> | ||
+ | |||
+ | |||
+ | <center> | ||
+ | <table class="wikitable sortable" border="0" style="text-align:left" width="600px" > | ||
+ | |||
+ | <caption align="top, left"> | ||
+ | </caption><tr bgcolor="#cccccc"> | ||
+ | <td> Name</td><td>Usage</td><td>Company</td></tr> | ||
+ | |||
+ | <tr> | ||
+ | <td><p> | ||
+ | <a href="http://www.nanodrop.com/Productnd2000overview.aspx">NanoDrop 2000 | ||
+ | </a> | ||
+ | </p></td><td>measurement of DNA concentration and purity </td><td>Thermo Scientific</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td><p> | ||
+ | <a href="http://catalog.bd.com/bdCat/viewProduct.doCustomer?productNumber=353046">6-well plate, polysterene | ||
+ | </a> | ||
+ | </p></td><td>light induction in UV-illumination chamber </td><td>BD, Sigma</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td><p> | ||
+ | <a href="http://catalog.bd.com/bdCat/viewProduct.doCustomer?productNumber=353047">24-well plate, polysterene | ||
+ | </a> | ||
+ | </p></td><td>light induction in UV-illumination chamber </td><td>BD, Sigma</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td><p> | ||
+ | <a href="http://www.sarstedt.com/php/main.php?newlanguage=en">Cuvettes | ||
+ | </a> | ||
+ | </p></td><td>visualization purposes </td><td>Sarstedt</td> | ||
+ | </tr> | ||
+ | |||
+ | |||
+ | <tr> | ||
+ | <td><p> | ||
+ | <a href="http://www.intas.de/geldokumentation-gel-ix-imager">Intas Gel IX Imager | ||
+ | </a> | ||
+ | </p></td><td>Gel Documentation and UV-illumination of Bl21(DE3) <i>E.coli</i> transformed with radiation sensing constructs; illumination wavelength: 312 nm </td><td>Intas</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td><p> | ||
+ | <a href="http://www.berthold.com/en/bio/product/mithras-lb-940-multimode-microplate-reader">Plate Reader | ||
+ | </a> | ||
+ | </p></td><td>ONPG Assay </td><td>Berthold Technologies</td> | ||
+ | </tr> | ||
+ | |||
+ | </table> | ||
+ | </center> | ||
+ | |||
+ | |||
+ | <p> </p> | ||
+ | </div><!--end SubWrapper--> | ||
+ | |||
+ | <div id="news"> | ||
+ | |||
+ | </div><!--end news--> | ||
+ | </div> <!-- end super_main_wrapper> | ||
+ | </body> | ||
</html> | </html> |
Latest revision as of 02:50, 17 June 2012
Kits for purification of plasmid-DNA and DNA fragments
DNA was extracted in small or medium scale using the Promega or Qiagen DNA preparation kits. For all DNA extractions before digestions/transformations, Promega Kits were applied. Only when performing sequencing, Qiagen kits were preferred.
Name | Product Description | Company |
small scale plasmid purification from E. coli Top10 cells before digestion or transformation into different E. coli strains | Promega | |
small scale plasmid purification from E. coli Top10 cells before sequencing | Qiagen | |
medium scale plasmid purification from E. coli Top10 cells before digestion or transformation into different E. coli strains | Promega | |
direct purification of DNA after restriction digests | Qiagen | |
Purification of DNA fragements from agarose gel | Qiagen |
LB growth media - components an recipe
LB media was prepared from 10 g/l tryptone, 5 g/l yeast extract and 5 g/l NaCl and autoclaved before usage. For preparing LB agar plates, 1.5 g of agar were added to the media. The media was furthermore substituted with 100 ug/ml ampicillin (for LB-amp) or 35 ug/ul chloramphenicol (for LB-cm).
Name | Usage | Company |
LB media | BD | |
LB media | BD | |
LB media | Sigma | |
preparing agar plates | Sigma | |
usage: 100 ug/ml, selection of plasmid containing an amp resistance gene | Sigma | |
usage: 35 ug/ml, selection of plasmid containing an Cm resistance gene | Sigma | |
preparing agar plates | Sigma | |
dissolve in DMSO at 20 mg/ml. final concentration for blue-white screening: 100 ug/ml and for X-Gal assays in liquid LB culture: 200 ug/ml | Sigma |
E. coli Strains
E. coli Top10 were used for amplification of plasmid DNA and subsequent DNA substraction. Bl21(DE3) were used in all measurments/assays performed.
Strain | Genotype |
F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(StrR) endA1 λ- | |
F– ompT gal dcm lon hsdSB(rB- mB-) λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) |
Enzymes
Name | Product Description | Company |
restriction enzyme, recognition sequence: GAATTC | Promega or NEB | |
restriction enzyme, recognition sequence: ACTAGT | Promega or NEB | |
restriction enzyme, recognition sequence: TCTAGA | Promega or NEB | |
restriction enzyme, recognition sequence: CTGCAG | Promega or NEB | |
Ligation of DNA fragments | Promega or Fermentas | |
Standard Taq PCR master mix for robust amplification of DNA-Fragments by PCR; used for colony-PCR screens | Fermentas |
Oligonucleotides
Oligos were annealed and subsequently cloned into EcoRI/XbaI predigested reporter backbones. Thereby short synthetic DNA-sequences (in this case for the promoters psulA, precA and precB) can be generated in a fast and easy manner.
Oligo Name | Oligo Sequence | sequence length |
psulA_fw | 5'-aattcgcggccgcttctagagGGGTTGATCTTTGTTGT CACTGGATGTACTGTACATCCATACAGTAACTCACc-3' | 74 |
psulA_rev | 5'-ctaggGTGAGTTACTGTATGGATGTACAGTACATCCAG TGACAACAAAGATCAACCCctctagaagcggccgcg-3' | 74 |
precB_fw | 5'aattcgcggccgcttctagagCCTGAAGGCTGGAAAGTGTGGGAGA ACGTCAGCGCGTTGCAGCAAACAATGCCCCTGATGAGTGAAAAGAc-3' | 92 |
precB_rev | 5'-ctaggTCTTTTCACTCATCAGGGGCATTGTTTGCTGCAACGCGCTG ACGTTCTCCCACACTTTCCAGCCTTCAGGctctagaagcggccgcg-3' | 92 |
precC_fw | 5'-aattcgcggccgcttctagagTTCACCCGGGGGCAGAGAAGGC GAGATGACCCGCCTGCATTGCCCGAATCGTCAGTAGTCAGGAGCCGCTc-3' | 92 |
precC_rev | 5'-ctaggAGCGGCTCCTGACTACTGACGATTCGGGCAATGCAGGCG GGTCATCTCGCCTTCTCTGCCCCCGGGTGAActctagaagcggccgcg-3' | 92 |
Gel electrophoresis
For gel electrophoresis we used 1 % agarose dissolved in 1x TAE buffer. Gels were run at 110 V for 25-60 min.
Name | Usage | Company |
to be dissolved to 1 % in 1x TAE buffer | Sigma | |
Tris-Acetate-EDTA buffer for gel electrophoresis | Sigma | |
dissolve to 1x in DNA samples before loading onto agarose gel | Promega | |
1kb DNA ladder | Promega | |
DNA ladder mix, 100 bp - 10 kb | Fermentas |
Measurements
The following materials and devices were used for our measurements and assays (DNA concentration, ONPG- and X-Gal assay)
Name | Usage | Company |
measurement of DNA concentration and purity | Thermo Scientific | |
light induction in UV-illumination chamber | BD, Sigma | |
light induction in UV-illumination chamber | BD, Sigma | |
visualization purposes | Sarstedt | |
Gel Documentation and UV-illumination of Bl21(DE3) E.coli transformed with radiation sensing constructs; illumination wavelength: 312 nm | Intas | |
ONPG Assay | Berthold Technologies |