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Team:TorontoMaRSDiscovery/Notebook/October
From 2009.igem.org
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Contents |
October 1, 2009
- Miniprepped Tet, 5, EncY, C
- Ran minipreps and K plasmid on a gel
- Besides Tet, all samples looked fine
- Poured K plates
October 2, 2009
- Digested K, EncY and ran on gel
October 3, 2009
- Digested EncY and old Tet plasmid -> ran on gel
- EncY looked fine, but Tet did not show up on gel
- Started overnight culture of Tet
October 4, 2009
- Miniprepped, digested Tet and ran on gel
- Tet was confirmed
- gel extracted EncY, Tet and got low yields
- Started overnight ligations
- 1: Digested Tet + EncY + 5
- 2: Gel extraced Tet + Digested EncY + 5
- negative controls were Tet plasmid alone
October 8, 2009
October 9, 2009
- Miniprepped EncY overnight and digested with E,P
- Ran digest on a gel
- Enc part confirmed
- Transfected Enc+5 ligations from Sunday/Monday into competent cells from Invitrogen
- Used NEB Hight Efficiency Transformation Protocol C2987
- did one 10-fold dilution
- transformed 5ul of DNA
- spread 100ul of cells on plate
- shaker/incubator speed 232rpm
- Used NEB Hight Efficiency Transformation Protocol C2987
October 11, 2009
- Checked plates, no colonies yet
- Picked colonies and started overnights from an old 12E plate that wasn't checked: 12E i,ii,iii
October 13, 2009
- 12E i and iii showed growth -> miniprepped
- Digested and ran on 1% agarose gel
- Stained gel for 1 hour in EtBr+buffer
- A faint ~700 band was observed! This could possibly be the 1+2+Enc insert that we want.
- To confirm the insert, we decided to sequence it
October 15, 2009
- Primers for sequencing 12E insert was designed and ordered
October 16, 2009
- EncY prepped and ready to ship down to MIT to submit part to registry
