Team:Heidelberg/Notebook/Material Methods
From 2010.igem.org
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Agarose Gel Electrophoresis<br> | Agarose Gel Electrophoresis<br> | ||
- | Agarose flat-bed gels in various concentrations (0.6–2% agarose in | + | Agarose flat-bed gels in various concentrations (0.6–2% agarose in 0.5 x TAE buffer) and sizes were run to separate DNA fragments in an electrical field (10–20 V/cm) for analytical or preparative use. The desired amount of agarose was boiled in 1 x TAE buffer until it was completely dissolved. After it cooled down to approximately 60°C, ethidium bromide (EtBr) solution (0.5 μg/ml final concentration) was added to the liquid agar, which was then poured in a flat-bed tray with combs. As soon as the agarose solidified, the Running buffer (0.5 x TAE buffer) was added before the DNA in the loading buffer was loaded into the wells and separated electrophoretically. Ethidium bromide intercalates with the DNA’s GC ntss resulting in DNA-EtBr-complex that emits visible light. Therefore, the DNA fragments could be detected on a UV-light tray at 265 nm. |
===Large scale preparation of plasmid DNA=== | ===Large scale preparation of plasmid DNA=== | ||
- | 150 ml LB-Medium with 150 µl ampicillin was inoculated with 50 µl of bacteria culture which grew overnight on a shaker at 37°C. The plasmid DNA was isolated using QiAprep Spin MAxiprep kit from Qiagen and the protocol was followed. The overnight culture was centrifuged for 20 min at 4000 rpm at 4°C using an SLA 1500 Rotor. Afterwards the LB-medium was discarded and the pellet was homogeneously resuspended in 10 ml of precooled Buffer P1. After having added 10 ml of Buffer P2 the mixture was inverted 4-6 times and incubated for 5 min at RT before adding 10 ml of chilled Buffer P3. Thereafter the lysate was poured into a prepared QIAfilter Maxi Cartridge and incubated at RT for 10 min. During this time a QIAGEN-tip 500 was equilibrated by applying 10 ml of Buffer QBT and allowing the column to empty by gravity flow. The cell lysate was filtered into the QIAGEN-tip. The cleared lysate entered the resin by gravity flow and after washing with 2 x 30 ml Buffer QC the Plasmid DNA was eluted with 15 ml Buffer QF. After this the DNA was precipitated by adding 10.5 ml isopropanol and centrifuged at 4,000 rpm for 45 min at 4°C. The supernatant was discarded and the DNA pellet was washed with 5 ml ethanol (70%) and centrifuged at 4,000 rpm for 15 min. After air-drying the pellet the DNA was redissolved in | + | 150 ml LB-Medium with 150 µl ampicillin was inoculated with 50 µl of bacteria culture which grew overnight on a shaker at 37°C. The plasmid DNA was isolated using QiAprep Spin MAxiprep kit from Qiagen and the protocol was followed. The overnight culture was centrifuged for 20 min at 4000 rpm at 4°C using an SLA 1500 Rotor. Afterwards the LB-medium was discarded and the pellet was homogeneously resuspended in 10 ml of precooled Buffer P1. After having added 10 ml of Buffer P2 the mixture was inverted 4-6 times and incubated for 5 min at RT before adding 10 ml of chilled Buffer P3. Thereafter the lysate was poured into a prepared QIAfilter Maxi Cartridge and incubated at RT for 10 min. During this time a QIAGEN-tip 500 was equilibrated by applying 10 ml of Buffer QBT and allowing the column to empty by gravity flow. The cell lysate was filtered into the QIAGEN-tip. The cleared lysate entered the resin by gravity flow and after washing with 2 x 30 ml Buffer QC the Plasmid DNA was eluted with 15 ml Buffer QF. After this the DNA was precipitated by adding 10.5 ml isopropanol and centrifuged at 4,000 rpm for 45 min at 4°C. The supernatant was discarded and the DNA pellet was washed with 5 ml ethanol (70%) and centrifuged at 4,000 rpm for 15 min. After air-drying the pellet the DNA was redissolved in H2O. <br> |
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Revision as of 10:41, 24 October 2010
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