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Team:Heidelberg LSL/Notebook materials
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| - | <h4>Kits for | + | <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> | <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> | ||
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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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<br> | <br> | ||
| - | <h4>E. coli Strains</h4> | + | <h4><i>E. coli</i> Strains</h4> |
| - | <p>E. coli Top10 were used for amplification of plasmid DNA and subsequent DNA substraction. Bl21(DE3) were used in all measurments/assays performed.</p> | + | <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> |
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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> |
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| - | <h4> | + | <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> | <p>For gel electrophoresis we used 1 % agarose dissolved in 1x TAE buffer. Gels were run at 110 V for 25-60 min.</p> | ||
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<a href="http://www.intas.de/geldokumentation-gel-ix-imager">Intas Gel IX Imager | <a href="http://www.intas.de/geldokumentation-gel-ix-imager">Intas Gel IX Imager | ||
</a> | </a> | ||
| - | </p></td><td>Gel Documentation and UV-illumination of Bl21(DE3) E. coli transformed with radiation sensing constructs; illumination wavelength: 312 nm </td><td>Intas</td> | + | </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> | ||
<tr> | <tr> | ||
<td><p> | <td><p> | ||
| - | <a href=" | + | <a href="http://www.berthold.com/en/bio/product/mithras-lb-940-multimode-microplate-reader">Plate Reader |
</a> | </a> | ||
</p></td><td>ONPG Assay </td><td>Berthold Technologies</td> | </p></td><td>ONPG Assay </td><td>Berthold Technologies</td> | ||
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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 |
