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Team:TU Munich/Notebook/Protocols
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''genomic DNA isolation from S.cerevisiae'' | ''genomic DNA isolation from S.cerevisiae'' | ||
| - | + | ''measurement of DNA concentration'' | |
| - | + | ''sequencing of plasmid DNA'' | |
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| + | ''Agarose gel-electrophoesis'' | ||
in vitro modification of DNA | in vitro modification of DNA | ||
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| - | + | ''Site-Directed Mutagenesis'' | |
| - | + | ''polymerase chain reaction (PCR)'' | |
| - | + | ''colony PCR'' | |
| - | + | ''purification of PCR products'' | |
| - | + | ''Dephosphorylation of DNA'' | |
| - | + | ''DNA restriction enzyme digest'' | |
| + | ''ligation / cycled ligation'' | ||
| + | ''Oligohybridization of single-stranded DNA'' | ||
| + | ''gene synthesis'' | ||
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o Aim of the experiment: Discrimination of growing ability in wort medium. | o Aim of the experiment: Discrimination of growing ability in wort medium. | ||
• Electroporation of electrocompetent E. coli with P698+699 | • Electroporation of electrocompetent E. coli with P698+699 | ||
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| + | =Materials= | ||
Revision as of 10:05, 13 September 2012
Methods
Molecular Biology Methods
plasmid DNA isolation from E.coli (miniprep) genomic DNA isolation from S.cerevisiae
measurement of DNA concentration sequencing of plasmid DNA
Agarose gel-electrophoesis in vitro modification of DNA
Site-Directed Mutagenesis polymerase chain reaction (PCR) colony PCR purification of PCR products Dephosphorylation of DNA DNA restriction enzyme digest ligation / cycled ligation Oligohybridization of single-stranded DNA gene synthesis
Protein biochemical methods
Protein expression in S.cerevisiae
Crude protein extraction from S.cerevisiae
SDS-Polyacrylamid-Gelelektrophoresis (SDS-PAGE)
Immunochemical Methods
Western Blot
Microbiological Methods
cultivation of E.coli
cultivation of S.cerevisiae
heat shock transformation of E.coli with plasmid DNA
transformation of S.cerevisiae
genome integration
Chemical Methods
Phycocyanobilin (PCB) extraction from dried Spirulina platensis powder
??? • Introducing new Saccharomyces cerevisiae strain (Y190 strain) from Schwab lab for Y2H o Aim of the experiment: The Y190 Saccharomyces cerevisiae is a special strain for Yeast-two-hybrid. This strain carries a Gal4 and Gal80 deletion to higher the signal/noise-ratio of protein-protein interactions. Reporter for protein-protein interactions are HIS3, lacZ and MEL1 and are encoded in the genomic DNA. Transformation markers are trp1, leu2 and cyhR2 and are encoded on the transformation plasmids. • Gradient PCR • Cycled ligation • Inoculation of YPD medium with S. cerevisiae and brewing yeast strain 34/70 (part 2/3) o Aim of the experiment: Discrimination of growing ability in wort medium. • Electroporation of electrocompetent E. coli with P698+699
