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Revision as of 17:51, 25 September 2012 (view source) KatrinFischer (Talk | contribs) (→DNA restriction enzyme digest) ← Older edit		Revision as of 17:52, 25 September 2012 (view source) KatrinFischer (Talk | contribs) (→purification of PCR products) Newer edit →
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	====Dephosphorylation of DNA====		====Dephosphorylation of DNA====

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Revision as of 17:52, 25 September 2012

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Methods

1. Molecular Biology Methods

Isolation of Plasmid DNA from E.coli (miniprep)

Plasmid DNA from E. coli was isolated from overnight cultures using the DNA extraction mini-prep kit (Qiagen).The principle of this method is the alkaline lysis of the bacterial cells followed by a selective immobilization of the plasmid DNA on a column, a couple of washing steps and the elution of the DNA.

Isolation of Genomic DNA from S.cerevisiae

Determination 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 plasmids (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

PCR products were purified using the PCR purification kit by Qiagen.

Dephosphorylation of DNA

DNA restriction enzyme digest

For the preparation of DNA fragments and the restriction analysis of plasmid DNA, the DNA was cut using restriction endonucleases. For restriction digestion, buffer and DNA concentrations were used according to the suggestions by the manufacturer.

ligation / cycled ligation

oligohybridization of single-stranded DNA

Site-Directed Mutagenesis

sequencing of plasmid DNA

DNA constructs were sequenced by [http://eurofinsdna.com/ Eurofins mwg operon] using our own sequencing primers.

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

Before transformation, CaCl₂ competent cells were created after Cohen et al., 1972. For the creation of competent cells, 50 ml LB medium were inoculated with an overnight culture of the used ‘’E.coli’’ strain and incubated at 37 °C, 180 rpm. After an OD ₅₅₀ of 0,5 was reached, the culture was centrifuged for 4 minutes at 5000 g for 10 minutes. The pellet was then resuspended in in 40 ml pre-chilled in 0,1 M MgCl₂ solution, centrifuged again and then again resuspended in 20 ml of pre-chilled 0,05 M CaCl₂ solution. After 30 minutes of incubation on ice, the cells were centrifuged again and resuspenden in 2 ml 0,05 M CaCl₂ solution, 15 % v/v glycerol. The competent cells were aliquoted and stored an – 80 °C. For the transformation, 100 µl competent cells and 1 ng plamid or 5 µg of a ligation product were mixed and incubated for 30 minutes on ice. Afterwards, the cells were heat shocked at 37 °C for 5 minutes, then mixed with 2 ml LB medium and incubated at 180 rpm and 37 °C for 30-45 minutes. The transformed cells were then plated on LB medium containing an antibiotic.

transformation of S.cerevisiae

genome integration

4. Chemical Methods

Phycocyanobilin (PCB) extraction from dried Spirulina platensis powder

5. Brewing

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)]