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	====plasmid DNA isolation from ''E.coli'' (miniprep)====		====plasmid DNA isolation from ''E.coli'' (miniprep)====
		+	Plasmid DNA from E. coli was isolated from over night cultures using the DNA extraction mini-prep kit (Qiagen). (see protocol)
		+
		+
	====genomic DNA isolation from ''S.cerevisiae''====		====genomic DNA isolation from ''S.cerevisiae''====
-	====measurement of DNA concentration====
		+
		+	====measurement of DNA concentration====
		+	DNA concentration was measured using a NanoDrop Spectrophotometer by Thermo Scientific. The concentration was calculated after determination of the specific absorbance of DNA at 260 nm. Furthermore, the ratio of sample absorbance at 260 and 280 nm and at 260 an 230 nm were measured to specify the purity of the samples. A ratio of 260/280 of ~1.8 is generally accepted as “pure” for DNA. If the ratio is appreciably lower, it may indicate contemination with protein, phenol or other substances that absorb strongly at or near 280 nm. The ratio of sample absorbance at 260 and 230 nm. This is a secondary measure of nucleic acid purityad is often higher than the respective 260/280 values. They are commonly in the range of 1.8-2.2.
	====Agarose gel-electrophoresis====		====Agarose gel-electrophoresis====
-	=====analytical gel-electrophoresis=====	+	To separate double-stranded DNA fragments by length, agarose gel-electrophoresis using ethidium bromide as a nucleic acid stain was applied (Sambrook et al., 1989). This method was used for the restriction analysis of plasmid  (analytical gel-electrophoresis) as well as for the isolation of DNA fragments (preparative gel-electrophoresis). After preparative gel-electrophoresis, the bands were cut out and purified using a Qiagen Gel extraction kit.
-	=====preparative gel-electrophoresis and gel-extraction=====	+

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Revision as of 12:42, 13 September 2012

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Contents 1 Methods 1.1 1. Molecular Biology Methods 1.1.1 plasmid DNA isolation from E.coli (miniprep) 1.1.2 genomic DNA isolation from S.cerevisiae 1.1.3 measurement of DNA concentration 1.1.4 Agarose gel-electrophoresis 1.1.5 polymerase chain reaction (PCR) 1.1.5.1 colony PCR 1.1.5.2 genomic PCR 1.1.5.3 purification of PCR products 1.1.6 Dephosphorylation of DNA 1.1.7 DNA restriction enzyme digest 1.1.8 ligation / cycled ligation 1.1.9 oligohybridization of single-stranded DNA 1.1.10 Site-Directed Mutagenesis 1.1.11 sequencing of plasmid DNA 1.1.12 gene synthesis 1.2 2. Protein Biochemical Methods 1.2.1 Protein expression in S.cerevisiae 1.2.2 Crude protein extraction from S.cerevisiae 1.2.3 SDS-Polyacrylamid-Gelelektrophoresis (SDS-PAGE) 1.2.4 Western Blot 1.3 3. Microbiological Methods 1.3.1 cultivation of E.coli 1.3.2 cultivation of S.cerevisiae 1.3.3 heat shock transformation of E.coli with plasmid DNA 1.3.4 transformation of S.cerevisiae 1.3.5 genome integration 1.4 4. Chemical Methods 1.4.1 Phycocyanobilin (PCB) extraction from dried Spirulina platensis powder 1.5 5. Brewing 2 Materials 2.1 Bacteria and yeast strains 2.2 used Plasmids 2.3 Reagents 2.4 Buffer and Solutions 2.5 Microbial Media 2.6 References:

Methods

1. Molecular Biology Methods

plasmid DNA isolation from E.coli (miniprep)

Plasmid DNA from E. coli was isolated from over night cultures using the DNA extraction mini-prep kit (Qiagen). (see protocol)

genomic DNA isolation from S.cerevisiae

measurement of DNA concentration

DNA concentration was measured using a NanoDrop Spectrophotometer by Thermo Scientific. The concentration was calculated after determination of the specific absorbance of DNA at 260 nm. Furthermore, the ratio of sample absorbance at 260 and 280 nm and at 260 an 230 nm were measured to specify the purity of the samples. A ratio of 260/280 of ~1.8 is generally accepted as “pure” for DNA. If the ratio is appreciably lower, it may indicate contemination with protein, phenol or other substances that absorb strongly at or near 280 nm. The ratio of sample absorbance at 260 and 230 nm. This is a secondary measure of nucleic acid purityad is often higher than the respective 260/280 values. They are commonly in the range of 1.8-2.2.

Agarose gel-electrophoresis

To separate double-stranded DNA fragments by length, agarose gel-electrophoresis using ethidium bromide as a nucleic acid stain was applied (Sambrook et al., 1989). This method was used for the restriction analysis of plasmid (analytical gel-electrophoresis) as well as for the isolation of DNA fragments (preparative gel-electrophoresis). After preparative gel-electrophoresis, the bands were cut out and purified using a Qiagen Gel extraction kit.

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)]