Team:TorontoMaRSDiscovery/Notebook/September



=September 1, 2009=
 * 1) only 3+2a overnight showed growth
 * 2) Miniprepped 3+2a
 * 3) digested 3+2a, 4+5R, 3, 4, 5 with E,P
 * 4) Transfected EncC+5 and negative control into DH-5 cells, plated on Tet plates
 * 5) mistakenly tranfected T, K plasmid into DH-5 cells (ccdb gene)
 * 6) Ran digests on a gel
 * 7) *3+2 still not confirmed, but all 4+5 samples showed expected band

=September 2, 2009=
 * 1) Transfected Tet, K (RFP) backbone plasmids and Amp, C (ccdb) plasmids into DH-5alpha cells and DB3.1 cells respectively and plated on appropriate antibiotic plates
 * 2) Enc+5 showed larger colony -> started overnight

=September 3, 2009=
 * 1) only 1+2 (1), (4) showed growth in C
 * 2) Miniprepped Enc+5, 1+2 (1), (4)
 * 3) Digested Enc+5 with E,P, 1+2 (1), (4) with E,S, 3+2a with X,P for 1.5h
 * 4) Ran digests and old Tet digest from Monday on a gel
 * 5) *Forgot to add plasmid to controls
 * 6) *Tet digest still good
 * 7) *Enc+5 digest doesn't have ~900bp band -> not confirmed
 * 8) Started overnight ligation of 1+2+3+2/Tet
 * 9) Started overnight cultures: Ampx1, Kx2 Enc+5 x3, Tet, BB7

=September 4, 2009=
 * 1) Miniprepped overnight cultures
 * 2) stocked Amp, K1, K2, and Enc+5 (2), (3), (4)
 * 3) Digested Enc+5 (1)-(4), K1,2, BB7
 * 4) Transfected 1+2+3+2 into DH-5 cells (50ul)
 * 5) *left on ice for 2.5h after adding ligation product
 * 6) *incubated on shaker for about 2h
 * 7) Started overnight of C plasmid (ccdb)

=September 5, 2009=
 * 1) Ran a gel of digests from yesterday with controls
 * 2) Miniprepped C overnight culture
 * 3) Tet plate (Tet with RFP) showed 2 red colonies

=September 7, 2009=
 * 1) Digested C plasmid and redigested Enc+5 (1), (3), K1, K2
 * 2) Started Tet1, Tet2 overnight culture

=September 8, 2009=
 * 1) Ran a gel of the digests
 * 2) *Enc+5 band still not showing may have to religate
 * 3) *K and C plasmids digests showed inserts
 * 4) Started log phase culture for Tet1, Tet2
 * 5) 1+2+3+2 plate still hasn't shown any colonies
 * 6) Digested 1+2, 3+2, Enc for assembly
 * 7) Ran digests on a gel
 * 8) Started overnight ligation of 1+2+Enc, 1+2+3+2 in K1

=September 9, 2009=
 * 1) Transfected cells with overnight ligations
 * 2) *2 samples per ligation
 * 3) *Plated and grew overnight at 37C

=September 10, 2009=
 * 1) Saw lots of growth on plates (no red colonies yet)
 * 2) Prepared 10 overnight growth placed in incubator at 30C in K antibiotics
 * 3) put plates back in incubator at 37C

=September 11, 2009=
 * 1) Overnight cultures (x5) were miniprepped

=September 12, 2009=
 * 1) Started 20h digest of miniprepped samples

=September 13, 2009=
 * 1) Ran digests and negative controls on gel, but no bands at all were seen.

=September 14, 2009=
 * 1) Started overnight cultures of same 5 samples

=September 15, 2009=
 * 1) only 1232 (3) showed growth, miniprepped
 * 2) started overnight cultures of for 1232 (2), (4), 12E (2) and EncY

=September 16, 2009=
 * 1) Only EncY showed growth, miniprepped
 * 2) picked streaks of cells from 12E plates and 1232 plates (2 streaks per plate) and started overnights along with controls
 * 3) *Target cells: 1232 mix1, 1232 mix2, 12E mix1, 12E mix2
 * 4) *Controls:
 * 5) **positive control: no antibiotics
 * 6) **negative control: Kanamycin and LB
 * 7) **control control: LB only

=September 17, 2009=
 * 1) 1232 mix1, 2 and 12E mix2 showed growth
 * 2) PCRed EncY plasmid and ran on gel
 * 3) *a huge ~700bp band was seen
 * 4) Prepared EncY for sequencing

=September 18, 2009=
 * 1) Digested 1232 (3), 1+2, 7, EncY, K
 * 2) Ran digests on gel
 * 3) *7 did not show up on gel
 * 4) *Enc band was faint/did not show up

=September 19, 2009=
 * 1) 12E replates showed some colonies -> started overnights: 12E a,b,c,d,e
 * 2) started overnight culture for EncY
 * 3) 1232 replates showed many colonies:
 * 4) *1232 (1) had all pink colonies (does NOT contain insert)
 * 5) *1232 (2) had no pink colonies (contains insert)
 * 6) *These plates were stored in the fridge

=September 20, 2009=
 * 1) Miniprepped overnights except 12E (which did not show any growth in LB with antibiotics)
 * 2) Digested 7,K, EncY, 12E a,b,d,e
 * 3) Ran digests on gel
 * 4) *only Enc was confirmed on this gel
 * 5) Started K1 overnight

=September 24, 2009=
 * 1) Made a new batch of chemically competent cells
 * 2) *poured cells into 50ml falcon tubes
 * 3) *350ul aliquots + 350ul 40% glycerol
 * 4) Started 12E overnights from transfections done yesterday
 * 5) *Named: 1-5, a-e, but these have been used already, hence 1-5 were renamed 6-10, a-e renamed f-j
 * 6) Transformed ligation from Monday and negative control into new cells and plated on new K plates

=September 25, 2009=
 * 1) Miniprepped 12E 6-10, f-j
 * 2) *overnights from 9,10,g,i had almost no growth (confirmed after 1st centriugation step: did not miniprep)
 * 3) Digested miniprepes and ran on gel -> no bands
 * 4) Started overnights again

=September 26, 2009=
 * 1) Only 12E (6),f,j,h
 * 2) Digested minipreps
 * 3) Ran a gel

=September 28, 2009=
 * 1) Started 5ml overnight cultures of 6,f,j,h with kanamycin
 * 2) *added 5ul in 25ml LB then separated into 4 aliquots (1 aliquot left over)

=September 29, 2009=
 * 1) took 400ul aliquots of the 4 samples for storage as stocks
 * 2) Miniprepped the rest of the samples
 * 3) Ran gel saw no colonies
 * 4) Rechecked plates: lack of red colonies is suspicious
 * 5) *DH5 may be somewhat resistant to K (suggested by Calvin)
 * 6) May need to gel extract backbone and parts before ligation
 * 7) We are out of K plates; need to make more