Difference between revisions of "Team:LMU-TUM Munich/Methods"
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| − | + | ==Preparation of plasmid DNA== | |
For transformation of E. coli XL-1 blue chemically competent cells, cells were thawed on ice for 5 mins. 50-100 ng plasmid DNA (or 200 ng ligation product) were added, and cells were further incubated on ice for 20-30 mins. Afterwards, a heatshock was applied at 42°C for 45 secs. Cells were then further incubated on ice for 2 mins, and 950 μl of LB medium were added. Cells were then shaken at 200 rpm for at least 1 h at 37 C, and plated on LB-agar plates containing the appropriate antibiotic. The next day, single clones were picked and used to inoculate a liquid culture of 5 ml LB medium. After being incubated overnight shaking (200 rpm) at 37°C, cells were spun down, and plasmid DNA was extracted using the Qiagen QIAprep Spin Miniprep Kit. If ligation products were used for transformation, analytical digestion of DNA was performed by digestion with suitable restriction enyzmes for 1 h at 37°C and the correct incorporation of the desired fragment was verified via analytical gel electrophoresis. Additionally, plasmids were sequenced using the Eurofins Genomics sequencing service. | For transformation of E. coli XL-1 blue chemically competent cells, cells were thawed on ice for 5 mins. 50-100 ng plasmid DNA (or 200 ng ligation product) were added, and cells were further incubated on ice for 20-30 mins. Afterwards, a heatshock was applied at 42°C for 45 secs. Cells were then further incubated on ice for 2 mins, and 950 μl of LB medium were added. Cells were then shaken at 200 rpm for at least 1 h at 37 C, and plated on LB-agar plates containing the appropriate antibiotic. The next day, single clones were picked and used to inoculate a liquid culture of 5 ml LB medium. After being incubated overnight shaking (200 rpm) at 37°C, cells were spun down, and plasmid DNA was extracted using the Qiagen QIAprep Spin Miniprep Kit. If ligation products were used for transformation, analytical digestion of DNA was performed by digestion with suitable restriction enyzmes for 1 h at 37°C and the correct incorporation of the desired fragment was verified via analytical gel electrophoresis. Additionally, plasmids were sequenced using the Eurofins Genomics sequencing service. | ||
| − | + | ==Determination of DNA concentrations== | |
| + | DNA concentrations were determined spectrophotometrically using a Nanodrop device. From the measured absorption at 260 nm, DNA concentrations were determined by considering an optical density of 1 equal to a concentration 50 ng/myl DNA. Furthermore, wavelengths at 280 nm and 230 nm were measured to confirm sample purity without contamination of proteins (280 nm) or carbohydrates, phenol or EDTA. Ratios of 260/280 nm being in the range between 1.6 and 2.0, as well as the ratios of 260/230 nm being between 2.0 and 2.2 generally indicate sample purity. | ||
| + | == | ||
| − | + | ==DNA digestion and ligation== | |
| − | |||
| − | |||
| − | + | ==Agarose gel electrophoresis== | |
| + | ==Polymerase chain reaction== | ||
| + | ==Mammalian cell culture== | ||
| − | + | ==Luciferase assay for the quantification of expression levels== | |
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | {{LMU-TUM_Munich_html_end}} | ||
Revision as of 08:05, 12 October 2016
Seitenverantwortliche/r: Jan\
Contents
Preparation of plasmid DNA
For transformation of E. coli XL-1 blue chemically competent cells, cells were thawed on ice for 5 mins. 50-100 ng plasmid DNA (or 200 ng ligation product) were added, and cells were further incubated on ice for 20-30 mins. Afterwards, a heatshock was applied at 42°C for 45 secs. Cells were then further incubated on ice for 2 mins, and 950 μl of LB medium were added. Cells were then shaken at 200 rpm for at least 1 h at 37 C, and plated on LB-agar plates containing the appropriate antibiotic. The next day, single clones were picked and used to inoculate a liquid culture of 5 ml LB medium. After being incubated overnight shaking (200 rpm) at 37°C, cells were spun down, and plasmid DNA was extracted using the Qiagen QIAprep Spin Miniprep Kit. If ligation products were used for transformation, analytical digestion of DNA was performed by digestion with suitable restriction enyzmes for 1 h at 37°C and the correct incorporation of the desired fragment was verified via analytical gel electrophoresis. Additionally, plasmids were sequenced using the Eurofins Genomics sequencing service.
Determination of DNA concentrations
DNA concentrations were determined spectrophotometrically using a Nanodrop device. From the measured absorption at 260 nm, DNA concentrations were determined by considering an optical density of 1 equal to a concentration 50 ng/myl DNA. Furthermore, wavelengths at 280 nm and 230 nm were measured to confirm sample purity without contamination of proteins (280 nm) or carbohydrates, phenol or EDTA. Ratios of 260/280 nm being in the range between 1.6 and 2.0, as well as the ratios of 260/230 nm being between 2.0 and 2.2 generally indicate sample purity.
==
DNA digestion and ligation
Agarose gel electrophoresis
Polymerase chain reaction
Mammalian cell culture
Luciferase assay for the quantification of expression levels
