Team:TU Munich/Notebook/Protocols
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= Methods = | = Methods = | ||
==1. Molecular Biology Methods== | ==1. Molecular Biology Methods== | ||
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====plasmid DNA isolation from ''E.coli'' (miniprep)==== | ====plasmid DNA isolation from ''E.coli'' (miniprep)==== | ||
====genomic DNA isolation from ''S.cerevisiae''==== | ====genomic DNA isolation from ''S.cerevisiae''==== | ||
====measurement of DNA concentration==== | ====measurement of DNA concentration==== | ||
- | + | ||
+ | |||
====Agarose gel-electrophoresis==== | ====Agarose gel-electrophoresis==== | ||
=====analytical gel-electrophoresis===== | =====analytical gel-electrophoresis===== | ||
=====preparative gel-electrophoresis and gel-extraction===== | =====preparative gel-electrophoresis and gel-extraction===== | ||
- | + | ||
====polymerase chain reaction (PCR)==== | ====polymerase chain reaction (PCR)==== | ||
=====colony PCR===== | =====colony PCR===== | ||
=====genomic PCR===== | =====genomic PCR===== | ||
=====purification of PCR products===== | =====purification of PCR products===== | ||
+ | |||
====Dephosphorylation of DNA==== | ====Dephosphorylation of DNA==== | ||
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====ligation / cycled ligation==== | ====ligation / cycled ligation==== | ||
====oligohybridization of single-stranded DNA==== | ====oligohybridization of single-stranded DNA==== | ||
+ | ====Site-Directed Mutagenesis==== | ||
+ | ====sequencing of plasmid DNA==== | ||
====gene synthesis==== | ====gene synthesis==== | ||
Revision as of 10:41, 13 September 2012
Methods
1. Molecular Biology Methods
plasmid DNA isolation from E.coli (miniprep)
genomic DNA isolation from S.cerevisiae
measurement of DNA concentration
Agarose gel-electrophoresis
analytical gel-electrophoresis
preparative gel-electrophoresis and gel-extraction
polymerase chain reaction (PCR)
colony PCR
genomic PCR
purification of PCR products
Dephosphorylation of DNA
DNA restriction enzyme digest
ligation / cycled ligation
oligohybridization of single-stranded DNA
Site-Directed Mutagenesis
sequencing of plasmid DNA
gene synthesis
2. Protein Biochemical Methods
Protein expression in S.cerevisiae
Crude protein extraction from S.cerevisiae
SDS-Polyacrylamid-Gelelektrophoresis (SDS-PAGE)
Western Blot
3. 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
4. Chemical Methods
Phycocyanobilin (PCB) extraction from dried Spirulina platensis powder
5. Brewing
??? • 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. • 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
Materials
Bacteria and yeast strains
used Plasmids
Reagents
Buffer and Solutions
Microbial Media
References:
- [http://www.ncbi.nlm.nih.gov/pubmed/20010584 Pubmed: Prasher, 1995: Using GFP to see the light.(Review)]