Team:TorontoMaRSDiscovery/Notebook/Augst

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Contents

August 1, 2009

  1. Overnight encapsulin cultures 1 and 6 (randomly selected) and the negative control were miniprepped
  2. The rest of the encapsulin cultures were stocked with 20% glycerol
  3. 6 digestions were done: Enc1, Enc6, Enc -ve, BB5, BB7, and C
    • 16ul of BB5 plasmid was used
    • 500ng of plasmid were used for the others
  4. The digestions were run on a 1.3% agarose gel in TAE
    • BB5 was confirmed and all other parts were correct as well
  5. Overnight ligation of 7+Enc in the PCR machine

August 2, 2009

  1. Miniprepped overnight cultures of 1+2 (Sample 3 and 5) and 3+2 (Sample 1 and 2)
  2. Digested these plasmid samples and ran on gel with negative control (straight from fridge)
    • The 3kbp in the digest of 1+2 Sample 5 was not expected
    • Comparing the 2 samples of 3+2, the digest of sample 1 did not have a 700bp band, and the undigested sample 1 was about 500bp shorter than the undigested sample 2. We suspect the plasmid in sample 1 did not take up the 3+2 part and somehow recircularized.
  3. Transfected overnight ligations of 7+Enc into DH-5alpha cells and plated onto C plates

August 3, 2009

  1. No colonies were found on 7+Enc plates

August 4, 2009

  1. Started overnight cultures of BB1+2 sample 1,2, 4 and K stocks
  2. Digested BB4, BB5 and C plasmid, and ran them on a gel
    • All bands were of expected sizes.
  3. Started overnight ligation of 4+5 into C plasmid

August 5, 2009

  1. Transfected overnight ligation and plated them on C plates
  2. Plated new C plates (probably meant poured)
  3. Miniprepped overnight cultures of BB1+2 and K plasmid

August 6, 2009

  1. Digested BB1+2 Samples 1,2,4 and K plasmid with restriction enzymes EcoR1 and Pst1
  2. The digests along with negative controls were ran on a gel
    • The 1+2 insert was not seen on the gel, probably because it is too small
    • The ccdb gene (~600bp) was not seen on the gel

August 7, 2009

  1. Started overnight cultures of 1+2 sample 1,2,4
  2. Retransformed BB1 using the iGEM 2007 Toronto team's protocol (heat shock for 90s) and plated on Amp plates
  3. Plated DH5alpha cells as a control on plain LB plates
      • This is thumotoga maritime genomic DNA for purpose of re-cloning
  4. Microcentrifuge tubes 1 and 2 placed in -20 freezer

August 8, 2009

  1. BB1 transformants showed many colonies
    • plates (a plate full of colonies)
    • -> increasing heat shock to 90s increased efficiency (Note: 3x DNA used in this transformation)
    • Calvin mentioned he does heat shock for 120s
    • Calvin also suggested to increase tranformation time, ie. after adding DNA, hold on ice for an hour
  2. DH-5alpha replates all grew on the plates, demonstrating LB can be re-autoclaved and poured as plates
    • there was no difference between transformants directly plated after adding LB+glucose or incubating for an hour
  3. Miniprepped overnights (1+2) samle 1,2,4, and K

August 10, 2009

  1. Digested plasmid samples miniprepped on Sat (1,2,4,K) and Enc, C plasmid with EcoR1 and Pst1
  2. Ran a gel for the digests
    • (1+2) Sample 1 appeared to have a ~100bp band
    • K plasmid digest did not show the ccdb gene (~700bp) - will start another culture with antibiotics
  3. Started overnight cultures of (1+2) Sample 1

August 11, 2009

  1. Digested 4, 5, Enc, C(E+P), C(X+S)
    • BB4 did not digest
    • 5, C is stored in fridge
  2. Calvin suggested increasing the concentration of enzyme and increase time of digestion due to thawing enzymes over weekend in fridge
  3. Miniprepped 1+2(1) with new kit, got great yield ~100ng/ul
  4. Gel extracted C (X+S) after it was CIPed
  5. Overnight ligation of Enc+C in PCR machine
  6. Calvin gave us 2 vials of RbCl cells in -80C
    • one time use only
    • thaw and add DNA

August 12, 2009

  1. Transformed Enc+C ligations
  2. Digested BB4 again
  3. Started overnight ligation of 4+5
  4. started overnight culture of BB7
  5. Problem was found in C plates: too soft
    • not enough agar?

August 14, 2009

  1. Miniprepped 5, 7, C with normal protocol of BioBasics Kit
    • Results were low ~30ng/ul
  2. Digested C and CIPed it
  3. Ran 4 digest and plasmid samples of 5, 7, C and the CIPed C plasmid samples of 5, 7, C and the CIPed C plasmid
  4. Gel extracted CIPed C plasmid
  5. Ligated 4+5 and Enc into C plasmid and plated

August 16, 2009

  1. Started overnight culture for BB5, 7, C

August 17, 2009

  1. Miniprepped overnight cultures of 5, 7, C
    • 7 sample had a very low yield
    • started another overnight for & and will miniprep again tomorrow
  2. Digested (1+2) Sample 1, (3+2) Sample 2, 4, 5 and K plasmid
  3. Started an overnight ligation for (1+2)+(3+2) and 4+5 in K plasmid
  4. Ran digests on a gel

August 18, 2009

  1. Tranfected ligation products into DH-5alpha cells and plated on old plates
  2. Miniprepped overnight culture of 7
  3. Digested Enc6, C (E,P)
  4. ran a gel with digests
  5. Digested Enc 6, C (X,S)
  6. Ran a gel with digests
  7. Gel extracted C (CIPed)
  8. Started overnight cultures for Tet, K plasmid
  9. Started overnight digest for Enc6

August 19, 2009

  1. Miniprepped overnight cultures of Tet, K
  2. Ran a gel of the ligations and the overnight digest
  3. Started overnight culture of 4+5 colonies
  4. Ligated Enc into CIPed C backbone
  5. Started overnight K and Tet digestion
  6. Poured Tet plates

August 20, 2009

  1. Digested C plasmid
  2. Ran gel of C, K, Tet digests
  3. Miniprepped 4+5 overnight cultures -> 4+5?
  4. Started overnight cultures of Tet and a negative control

August 21, 2009

  1. No growth of negative Tet overnight culture
  2. Tet overnights were miniprepped
  3. Digested 4+5?, Tet, and TetX with (EcoR1 and Pst1)
  4. The digests were run on a gel with negative controls
    • None of the samples were confirmed
  5. Started overnights for Tet plasmid, 2 with tetracyclin and 1 with ampicillin

August 22, 2009

  1. Miniprepped overnights:
    • Tet1: Did 2 minipreps, one with normal protocol, one with low copy plasmid protocol
      • Normal protocol gave a higher yield
    • Tet with ampicillin: used low copy plasmid protocol, but cell pellet was much bigger therefore got the same yield as Tet1 normal protocol
  2. Digested the 2 samples with higher yields (500ng)
  3. Ran digests on gel, but still couldn't see ccdb gene
  4. Started to doubt restriction enzyme function, therefore redigested Tet H (1ug) and C plasmid (500ng)
  5. (1.5h digests for all digests today)
  6. Ran Tet H and C digests on a gel
    • A faint ~700bp band was observed in the Tet lane
    • More plasmid should be used for plasmid digests
  7. Started overnight ligation of 4+5 in Tet backbone

August 23, 2009

  1. Transfected 4+5, Enc+C (x2) ligations into 1 tube of Calvin's cells (aliquots of 33ul of cells)
  2. plated Enc transformants onto C plates
  3. Since there were no Tet plates, 400ul of tetracyclin was spread onto a plain LB plate and dried, before plating 4+5 transformants
  4. Started overnight cultures of 1+2 and 3+2 in 5ml of LB

August 24, 2009

  1. Replated left over cells
  2. Miniprepped 1+2 overnights, yielded ~40ng/ul
  3. 3+2(2) showed no growth
  4. Plates in the incubator showed no colonies

August 25, 2009

  1. Enc replate showed 3 colonies
  2. Started a log phase growth of 3+2(2), but it showed not growth again
  3. Started overnight cultures:
    • Enc A, B, C
    • 3+2 (2), (3), (4), (5), and a 3+2 (2) positive control (in LB only)
    • BB3 and BB4

August 26, 2009

  1. 4+5 plates showed 2 big colonies and 1 small colony
  2. Started log phase for 4+5 (1), (2) (did not stock)
  3. 3+2 overnights did not grow
    • They are not C-resistant?
    • We have to redo the 3+2 ligations
  4. Miniprepped Enc A, B, C, BB3 and BB4
  5. Digested miniprepped samples and also BB2, 1+2 plasmid for 2 hours
  6. Ran digests on a gel
    • 1ug of plasmid was digested for Enc A, B, C, BB2, BB3, BB4; and 1/5 was loaded on to gel (10ul of digest)
  7. 30x37.4=1122ng of plasmid was digested for 1+2; and 16.5ul was loaded (1122x16.5/50=370.26ng)
  8. Miniprepped log phase of 4+5 (1), (2)

August 27, 2009

  1. Digested 4+5 (1), (2), BB7 with E+P
  2. Ran digstes on gel
    • wall between lane 5 and 6 was broken upon loading
  3. religated BB3 digest and 3+2 into C plasmid for 2 hours at room temperature
  4. plated ligations: 2ul of ligation in 25ul of cells

August 28, 2009

  1. Transfected 50ul of DH5 cells with 4+5(1) plasmid (2ul)
  2. added 100ul of LB+glucose and plated all on a Tet plate
  3. Mixed up LB+agar for pouring C plates on Monday (Aug 31)
  4. Picked colonies growing on 4+5 plate and Enc plate and started overnight cultures

August 29, 2009

  1. Stocked Enc, 4+5 overnights and stored remaining culture in the fridge (4C)
  2. 4+5 retransfection plate did not show colonies

August 30, 2009

  1. 4+5 retransfection plate had full plate of colonies
  2. Overnight cultures of 4+5 and BB7 were started

August 31, 2009

  1. Stocked 4+5R culture (retransfection)
  2. Miniprepped overnight cultures of 4+5 (3,4,5,R), BB7, and EncX,Y,Z
  3. Digested EncC with E,S; 5 with X,P; Tet, 7, EncX,Y,Z with E,P
  4. Ran digests on a gel
    • EncY shows Enc insert
    • 5 digest lane shows a thick band the same size of the linear isoform in the control
  5. Poured C plates
  6. 3+2 tranformation from Aug 27 showed a few colonies
  7. 3 largest colonies were picked to start overnight culture
  8. Started overnight ligation of Enc+5/Tet with negative control
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