Team:Exeter/lab book/gibs/wk2
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Revision as of 09:36, 26 September 2012
Operon Construction: 16th - 20th July 2012 **Monday 16.7.12**TetrRBS and RBS (BBa_B0034) BioBricks resuspended and used for competent cell transformation. 1µl of resuspended DNA added to 25µl top10 competent cells. LB(amp) (1:1000) plates used for 20µl and 100µl spread plates. **Tuesday 17.7.12** Previous days spread plate colonies transferred to liquid medium, 1:1000 LB(amp) broth. **Wednesday 18.7.12** Centrifuged tubes, 4℃, 10 minutes, 3901 rcf Miniprep attempt from protocol Pellet was considered too small, believed to be due to shaking incubator turning off overnight 4 colonies were transferred to liquid medium as on previous day **Thursday 19.7.12** Transferred cultures to identical falcon tubes to remove pipette tips Centrifuged at 3901 rcf for 5 minutes, 4 °C Supernatant tipped away Miniprep attempt 2 Step 10 50 µl elution buffer added Incubated for 2 minutes at room temperature Centrifuged for 2 minutes, 13000 rpm 20 µl milli-Q water added Centrifuged for 2 minutes, 13000 rpm Columns discarded Eluted DNA put on ice for analysis with nanodrop spectrophotometer and then stored at -20 °C Plasmids from miniprep digested with EcoR1-HF and PstI according to digestion protocol
EcoR1-HF - 1 µl PstI - 1 µl 10x NEBuffer 2 - 5 µl 100x BSA - 0.5 µl MQ Water - 36 µl Incubated for 10 minutes at 37 °C DNA run on gel to test fragments |