Team:Heidelberg LSL/Notebook materials

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iGEM-2012HS - LSL-Heidelberg iGEM-2012HS - LSL-Heidelberg


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.


NameProduct DescriptionCompany

PureYield™ Plasmid Miniprep System

small scale plasmid purification from E. coli Top10 cells before digestion or transformation into different E. coli strains Promega

QIAprep Spin MiniPrep Kit (50)

small scale plasmid purification from E. coli Top10 cells before sequencing Qiagen

PureYield™ Plasmid Midiprep System

medium scale plasmid purification from E. coli Top10 cells before digestion or transformation into different E. coli strains Promega

QIAquick Nucleotide Removal Kit

direct purification of DNA after restriction digestsQiagen

QIAquick Gel Extraction Kit (50)

Purification of DNA fragements from agarose gelQiagen

Enzymes

NameProduct DescriptionCompany

EcoRI

restriction enzyme, recognition sequence: GAATTC Promega or NEB

SpeI

restriction enzyme, recognition sequence: ACTAGT Promega or NEB

PureYield™ Plasmid Midiprep System

restriction enzyme, recognition sequence: TCTAGAPromega or NEB

PstI

restriction enyzme, recognition sequence: CTGCAGPromega or NEB

T4 DNA ligase

Ligation of DNA fragmentsPromega or Fermentas

2x PCR MasterMix

Standard Taq PCR master mix for robust amplification of DNA-Fragments by PCR; used for colony-PCR screensFermentas

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 NameOligo Sequencesequence length
psulA_fw5'-aattcgcggccgcttctagagGGGTTGATCTTTGTTGT CACTGGATGTACTGTACATCCATACAGTAACTCACc-3'74
psulA_rev5'-ctaggGTGAGTTACTGTATGGATGTACAGTACATCCAG TGACAACAAAGATCAACCCctctagaagcggccgcg-3'74
precB_fw5'aattcgcggccgcttctagagCCTGAAGGCTGGAAAGTGTGGGAGA ACGTCAGCGCGTTGCAGCAAACAATGCCCCTGATGAGTGAAAAGAc-3'92
precB_rev5'-ctaggTCTTTTCACTCATCAGGGGCATTGTTTGCTGCAACGCGCTG ACGTTCTCCCACACTTTCCAGCCTTCAGGctctagaagcggccgcg-3'92
precC_fw5'-aattcgcggccgcttctagagTTCACCCGGGGGCAGAGAAGGC GAGATGACCCGCCTGCATTGCCCGAATCGTCAGTAGTCAGGAGCCGCTc-3'92
precC_rev5'-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).

NameProduct DescriptionCompany

Trypton

for preparing LB media BD

Yeast Extract

for preparing LB media BD

NaCl

for preparing LB media Sigma

Agar

for preparing agar plates Sigma

Ampicillin

usage: 100 ug/ml, selection of plasmid containing an amp resistance gene Sigma

Chloramphenicol

usage: 35 ug/ml, selection of plasmid containing an Cm resistance gene Sigma