Team:Heidelberg/Project/miRNA Kit
From 2010.igem.org
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- | Large scale preparation of plasmid DNA< | + | Large scale preparation of plasmid DNA<br> |
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 200 µl of H2O. 25 µg of plasmid rheb5’UTR-FLUC was digested with Ecl136II. Therefore 25 µg of plasmid rheb5'UTR-FLUC was mixed with 5 µl of 10 x Ecl136II buffer and 5 µl of Ecl136II and add ddH2O to a reaction volume of 50 µl. The mixture is incubated for 3 h at 37°C. To recover the DNA the sample was filled up to 100 µl by adding ddH2O. The same volume of phenol chloroform isoamyl alcohol was added and it was mixed thoroughly. Centrifugation was done for 5 min at 13,200 rpm. The aqueous phase was recovered and chloroform was added to extract the DNA again. The DNA was precipitated by adding one volume of isopropanol and mixing well. It was centrifuged for 20 min at 13,200 rpm and 4°C. The supernatant was carefully removed and the pellet was washed with 100 µl of 75% ethanol. It was centrifuged for 5 min at 13,200 rpm at 4°C. <br> | 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 200 µl of H2O. 25 µg of plasmid rheb5’UTR-FLUC was digested with Ecl136II. Therefore 25 µg of plasmid rheb5'UTR-FLUC was mixed with 5 µl of 10 x Ecl136II buffer and 5 µl of Ecl136II and add ddH2O to a reaction volume of 50 µl. The mixture is incubated for 3 h at 37°C. To recover the DNA the sample was filled up to 100 µl by adding ddH2O. The same volume of phenol chloroform isoamyl alcohol was added and it was mixed thoroughly. Centrifugation was done for 5 min at 13,200 rpm. The aqueous phase was recovered and chloroform was added to extract the DNA again. The DNA was precipitated by adding one volume of isopropanol and mixing well. It was centrifuged for 20 min at 13,200 rpm and 4°C. The supernatant was carefully removed and the pellet was washed with 100 µl of 75% ethanol. It was centrifuged for 5 min at 13,200 rpm at 4°C. <br> | ||
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Revision as of 01:44, 24 October 2010
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