From 2009.igem.org

Latest revision as of 18:16, 21 October 2009

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
   • 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
   • Forgot to add plasmid to controls
   • Tet digest still good
   • Enc+5 digest doesn't have ~900bp band -> not confirmed
5. Started overnight ligation of 1+2+3+2/Tet
6. 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)
   • left on ice for 2.5h after adding ligation product
   • incubated on shaker for about 2h
5. 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
   • Enc+5 band still not showing may have to religate
   • K and C plasmids digests showed inserts
2. Started log phase culture for Tet1, Tet2
3. 1+2+3+2 plate still hasn't shown any colonies
4. Digested 1+2, 3+2, Enc for assembly
5. Ran digests on a gel
6. Started overnight ligation of 1+2+Enc, 1+2+3+2 in K1

September 9, 2009

1. Transfected cells with overnight ligations
   • 2 samples per ligation
   • 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
   • Target cells: 1232 mix1, 1232 mix2, 12E mix1, 12E mix2
   • Controls:
     • positive control: no antibiotics
     • negative control: Kanamycin and LB
     • control control: LB only

September 17, 2009

1. 1232 mix1, 2 and 12E mix2 showed growth
2. PCRed EncY plasmid and ran on gel
   • a huge ~700bp band was seen
3. Prepared EncY for sequencing

September 18, 2009

1. Digested 1232 (3), 1+2, 7, EncY, K
2. Ran digests on gel
   • 7 did not show up on gel
   • 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:
   • 1232 (1) had all pink colonies (does NOT contain insert)
   • 1232 (2) had no pink colonies (contains insert)
   • 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
   • only Enc was confirmed on this gel
4. Started K1 overnight

September 24, 2009

1. Made a new batch of chemically competent cells
   • poured cells into 50ml falcon tubes
   • 350ul aliquots + 350ul 40% glycerol
2. Started 12E overnights from transfections done yesterday
   • Named: 1-5, a-e, but these have been used already, hence 1-5 were renamed 6-10, a-e renamed f-j
3. 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
   • overnights from 9,10,g,i had almost no growth (confirmed after 1st centriugation step: did not miniprep)
2. Digested miniprepes and ran on gel -> no bands
3. 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
   • 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
   • DH5 may be somewhat resistant to K (suggested by Calvin)
5. May need to gel extract backbone and parts before ligation
6. We are out of K plates; need to make more

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