From 2009.igem.org

Latest revision as of 18:16, 21 October 2009

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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