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Team:RMIT Australia/Notebook
From 2010.igem.org
Notebook
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20 AugustDid two PCR reaction and a transformation The first PCR we ran was a touch down PCR. Temperatures ranged from 69 to 64. The second PCR was a two step PCR. 30 AugustOrdered the TAQ polymerase from Mr. Gene. Should have it in 15 days. Meeting today at 10am to discuss project budget and costings. After the meeting we all are sitting together for a writing session. 31 AugustSpent the morning organising our flights In the afternoon we all sat together for a writing session 1 SeptemberWe all sat together for another writing session. 7 SeptemberHad a team meeting 14 SeptemberHad a team meeting and made a lab time table, we also set out tasks which need to be done by the next meeting
15 SeptemberMutagenesis Quikchange XL kit Click Here for Mutagenesis Protocol
We optimised for low 260/280 Reaction Mixture
First mutagenesis was to turn the -35 site in the promoter region into an Afi II restriction site. Dpn1 digest (2 µl) for 2hrs 16 SeptemberClick Here for Mutagenesis Protocol 2 µl of the mutagenesis product into 100 µl of competent cells Plate 50 µl, 100 µl and 150 µl samples. 17 September- finalized project abstract 19 September- Sent off project abstract to HQ 20 SeptemberTwo colonies were picked from the plates and were grown in 5mL of Media 21 SeptemberA miniprep was performed on 1.5ul of the overnight culture. 10ng/ul DNA were obtained from both samples with a O.D higher than 1.85. These samples were then used to perform a restriction digest using EcoR1 H.F and AflII. The restriction digest was done according to the iGEM protocols [http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf CLICK HERE]. Results The restriction digest did not work. We expected a band at 160bp and one at ~3000bp 22 SeptemberDNA sample ng/µl 1 µg Buffer (µl) EcoRI (µl) Afi II (µl) BSA (µl) Total H2O 1 80 12.5 4 1 1 0.5 19.0 6 2 70 14.3 4 1 1 0.5 20.8 4.2 3 66 15.2 4 1 1 0.5 21.7 3.3 4 80 12.5 4 1 1 0.5 19.0 6 5 63 16 4 1 1 0.5 22.5 2.5 6 56 17.9 4 1 1 0.5 24.4 0.6 7 106 9.4 4 1 1 0.5 15.9 9.1 8 94 10.6 4 1 1 0.5 17.1 7.9 9 94 10.6 4 1 1 0.5 17.1 7.9 10 88 11.4 4 1 1 0.5 17.9 7.1 11 119 8.4 4 1 1 0.5 14.9 10.1 12 123 8.1 4 1 1 0.5 14.6 10.4 Volume total of 25 µl Incubate at 37°C for 70 minutes
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