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Team:Calgary/29 July 2009
From 2009.igem.org
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Plasmid was isolated from an overnight culture of cl lamba using Sigma GenElute Mini Prep Kit. The concentration and purity of the isolated plasmid was then measured with the NanoDrop spectrophotometer. Restriction digest was performed with XbaI and PstI in React 2 (Invitrogen) buffer for 2 hours at 37ºC. Cut plasmid and uncut plasmid were then run on a 1.0% agarose gel, no bands were seen for either column. The isolated plasmid was then specked again, to verify the initial concentration, which turned out to be accurate. An overnight Restriction digest was then set up as described above; however, 600ng of DNA were used instead of 200ng. | Plasmid was isolated from an overnight culture of cl lamba using Sigma GenElute Mini Prep Kit. The concentration and purity of the isolated plasmid was then measured with the NanoDrop spectrophotometer. Restriction digest was performed with XbaI and PstI in React 2 (Invitrogen) buffer for 2 hours at 37ºC. Cut plasmid and uncut plasmid were then run on a 1.0% agarose gel, no bands were seen for either column. The isolated plasmid was then specked again, to verify the initial concentration, which turned out to be accurate. An overnight Restriction digest was then set up as described above; however, 600ng of DNA were used instead of 200ng. | ||
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A colony PCR was set up with colonies of the construction LuxPQ-B0015-R0040-LuxOU-B0015 in psB1AC3 as the template. Seven colonies were chosen. This PCR was performed with BBK CP F/R primers (these anneal on the BBK vector just outside the cloning site) and LuxPQ F / LuxOU R primers (these are gene specific primers). Due to the long extension time for this PCR (6min and 20 seconds) it is run overnight and then held at 4ºC upon completion. For PCR conditions, refer to PCR performed on July 29 2009. Restreaks of the seven colonies were made and overnight liquid cultures were made so that plasmid can be isolated tomorrow. | A colony PCR was set up with colonies of the construction LuxPQ-B0015-R0040-LuxOU-B0015 in psB1AC3 as the template. Seven colonies were chosen. This PCR was performed with BBK CP F/R primers (these anneal on the BBK vector just outside the cloning site) and LuxPQ F / LuxOU R primers (these are gene specific primers). Due to the long extension time for this PCR (6min and 20 seconds) it is run overnight and then held at 4ºC upon completion. For PCR conditions, refer to PCR performed on July 29 2009. Restreaks of the seven colonies were made and overnight liquid cultures were made so that plasmid can be isolated tomorrow. | ||
Revision as of 23:23, 29 July 2009
UNIVERSITY OF CALGARY
