"
Team:Heidelberg LSL/Notebook materials
From 2012hs.igem.org
| Line 101: | Line 101: | ||
<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> | ||
Revision as of 16:01, 11 June 2012
Kits for Plasmid and DNA purification
DNA was extracted in small or medium scale using the Promega or Qiagen DNA preparation kits. 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 |
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 enyzme, 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 |
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 | Product Description | Company |
| for preparing LB media | BD | |
| for preparing LB media | BD | |
| for preparing LB media | Sigma | |
| for 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 |
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 | Product Description | Company |
| to be dissolved to 1 % in 1x TAE buffer | Sigma | |
| for preparing LB media | BD | |
| 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 |
