Team:Heidelberg LSL/Notebook materials

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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
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 digests	Qiagen
QIAquick Gel Extraction Kit (50)	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
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
petri dish (10 cm)	preparing agar plates	Sigma
X-Gal	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
Top10	F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(StrR) endA1 λ-
Bl21(DE3)	F– ompT gal dcm lon hsdSB(rB- mB-) λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5])

Enzymes

Name	Product Description	Company
EcoRI	restriction enzyme, recognition sequence: GAATTC	Promega or NEB
SpeI	restriction enzyme, recognition sequence: ACTAGT	Promega or NEB
XbaI	restriction enzyme, recognition sequence: TCTAGA	Promega or NEB
PstI	restriction enyzme, recognition sequence: CTGCAG	Promega or NEB
T4 DNA ligase	Ligation of DNA fragments	Promega or Fermentas
2x PCR MasterMix	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
Agarose	to be dissolved to 1 % in 1x TAE buffer	Sigma
Yeast Extract	for preparing LB media	BD
TAE buffer	Tris-Acetate-EDTA buffer for gel electrophoresis	Sigma
Blue/Orange loading dye, 6x	dissolve to 1x in DNA samples before loading onto agarose gel	Promega
1kb DNA ladder	1kb DNA ladder	Promega
GeneRuler DNA ladder Mix	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
NanoDrop 200	measurement of DNA concentration and purity	Thermo Scientific
6-well plate, polysterene	light induction in UV-illumination chamber	BD, Sigma
24-well plate, polysterene	light induction in UV-illumination chamber	BD, Sigma
Cuvettes	visualization purposes	Sarstedt
Gel Documantation Chamber	Gel Documentation and UV-illumination of Bl21(DE3) E. coli transformed with radiation sensing constructs	Company Name
Plate Reader	ONPG Assay	Company Name