Team:TorontoMaRSDiscovery/Notebook/October



=October 1, 2009=
 * 1) Miniprepped Tet, 5, EncY, C
 * 2) Ran minipreps and K plasmid on a gel
 * 3) *Besides Tet, all samples looked fine
 * 4) Poured K plates

=October 2, 2009=
 * 1) Digested K, EncY and ran on gel

=October 3, 2009=
 * 1) Digested EncY and old Tet plasmid -> ran on gel
 * 2) *EncY looked fine, but Tet did not show up on gel
 * 3) Started overnight culture of Tet

=October 4, 2009=
 * 1) Miniprepped, digested Tet and ran on gel
 * 2) *Tet was confirmed
 * 3) gel extracted EncY, Tet and got low yields
 * 4) Started overnight ligations
 * 1: Digested Tet + EncY + 5
 * 2: Gel extraced Tet + Digested EncY + 5
 * 1) *negative controls were Tet plasmid alone

=October 8, 2009=

=October 9, 2009=
 * 1) Miniprepped EncY overnight and digested with E,P
 * 2) Ran digest on a gel
 * 3) *Enc part confirmed
 * 4) Transfected Enc+5 ligations from Sunday/Monday into competent cells from Invitrogen
 * 5) *Used NEB Hight Efficiency Transformation Protocol C2987
 * 6) **did one 10-fold dilution
 * 7) **transformed 5ul of DNA
 * 8) **spread 100ul of cells on plate
 * 9) **shaker/incubator speed 232rpm

=October 11, 2009=
 * 1) Checked plates, no colonies yet
 * 2) Picked colonies and started overnights from an old 12E plate that wasn't checked: 12E i,ii,iii

=October 13, 2009=
 * 1) 12E i and iii showed growth -> miniprepped
 * 2) Digested and ran on 1% agarose gel
 * 3) *Stained gel for 1 hour in EtBr+buffer
 * 4) *A faint ~700 band was observed! This could possibly be the 1+2+Enc insert that we want.
 * 5) To confirm the insert, we decided to sequence it

=October 15, 2009=
 * 1) Primers for sequencing 12E insert was designed and ordered

=October 16, 2009=
 * 1) EncY prepped and ready to ship down to MIT to submit part to registry