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Team:NRP-UEA-Norwich/Week9
From 2012.igem.org
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Contents |
. We looked at the plates of transformant colonies of RFP and GFP that have kanamycin resistance. Both Rebecca's and Rachel's plates for GFP looked a little dodgy. Rebecca's had not grown at all whereas, Rachel's had but the colonies were rather large. The RFP colonies looked good. However, after double checking in the parts registry, we realised that these BioBricks have no RBS and therefore, we decided to keep these plates for now and went back to the previous BioBricks used with RFP, GFP and CFP.
. Russell, Rebecca and Khadija used Pst1 and Xba1 to do a double restriction digest of RFP, GFP and CFP that we previously used. We decided that despite the backbone fragment seemingly being too large, we would try it out. Into each eppendorf a 3.2µl mastermix sample of 5µl Pst1, 5µl Xba1, 2µl of BSA ad 20µl Buffer H. There were 9 eppendorf tubes containing: 3.6µl of DNA and 13.2µl of water (CFP1), 6.03µl of DNA and 10.77µl of water (CFP2), 3.47µl of DNA and 13.32µl of water (RFP1), 3.92µl of DNA and 12.88µl of water (RFP2), 3µl of DNA and 13.8µl of water (GFP1), 3.91µl of DNA and 12.89µl of water (GFP3) and 3.74µl of DNA and 13.06µl of water (GFP4). These were left overnight.
. At the same time, as we were using isolated plasmid DNA we decided to transform the original DNA into alpha competent silver standard cells from Bioline.
Rachel purified using promega wizard kit and protocol RFP that had been cut from gel.
