Team:TorontoMaRSDiscovery/Notebook/Augst
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=August 1, 2009= | =August 1, 2009= |
Revision as of 20:39, 19 October 2009
Home | The Team | The Project | Parts Submitted to the Registry | Modeling | Bioinformatics | Notebook |
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August 1, 2009
- Overnight encapsulin cultures 1 and 6 (randomly selected) and the negative control were miniprepped
- The rest of the encapsulin cultures were stocked with 20% glycerol
- 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
- The digestions were run on a 1.3% agarose gel in TAE
- BB5 was confirmed and all other parts were correct as well
- Overnight ligation of 7+Enc in the PCR machine
August 2, 2009
- Miniprepped overnight cultures of 1+2 (Sample 3 and 5) and 3+2 (Sample 1 and 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.
- Transfected overnight ligations of 7+Enc into DH-5alpha cells and plated onto C plates
August 3, 2009
- No colonies were found on 7+Enc plates
August 4, 2009
- Started overnight cultures of BB1+2 sample 1,2, 4 and K stocks
- Digested BB4, BB5 and C plasmid, and ran them on a gel
- All bands were of expected sizes.
- Started overnight ligation of 4+5 into C plasmid
August 5, 2009
- Transfected overnight ligation and plated them on C plates
- Plated new C plates (probably meant poured)
- Miniprepped overnight cultures of BB1+2 and K plasmid
August 6, 2009
- Digested BB1+2 Samples 1,2,4 and K plasmid with restriction enzymes EcoR1 and Pst1
- 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
- Started overnight cultures of 1+2 sample 1,2,4
- Retransformed BB1 using the iGEM 2007 Toronto team's protocol (heat shock for 90s) and plated on Amp plates
- Plated DH5alpha cells as a control on plain LB plates
- This is thumotoga maritime genomic DNA for purpose of re-cloning
- This is thumotoga maritime genomic DNA for purpose of re-cloning
- Microcentrifuge tubes 1 and 2 placed in -20 freezer
August 8, 2009
- 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
- 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
- Miniprepped overnights (1+2) samle 1,2,4, and K
August 10, 2009
- Digested plasmid samples miniprepped on Sat (1,2,4,K) and Enc, C plasmid with EcoR1 and Pst1
- 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
- Started overnight cultures of (1+2) Sample 1
August 11, 2009
- Digested 4, 5, Enc, C(E+P), C(X+S)
- BB4 did not digest
- 5, C is stored in fridge
- Calvin suggested increasing the concentration of enzyme and increase time of digestion due to thawing enzymes over weekend in fridge
- Miniprepped 1+2(1) with new kit, got great yield ~100ng/ul
- Gel extracted C (X+S) after it was CIPed
- Overnight ligation of Enc+C in PCR machine
- Calvin gave us 2 vials of RbCl cells in -80C
- one time use only
- thaw and add DNA
August 12, 2009
- Transformed Enc+C ligations
- Digested BB4 again
- Started overnight ligation of 4+5
- started overnight culture of BB7
- Problem was found in C plates: too soft
- not enough agar?
August 14, 2009
- Miniprepped 5, 7, C with normal protocol of BioBasics Kit
- Results were low ~30ng/ul
- Digested C and CIPed it
- 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
- Gel extracted CIPed C plasmid
- Ligated 4+5 and Enc into C plasmid and plated
August 16, 2009
- Started overnight culture for BB5, 7, C
August 17, 2009
- Miniprepped overnight cultures of 5, 7, C
- 7 sample had a very low yield
- started another overnight for & and will miniprep again tomorrow
- Digested (1+2) Sample 1, (3+2) Sample 2, 4, 5 and K plasmid
- Started an overnight ligation for (1+2)+(3+2) and 4+5 in K plasmid
- Ran digests on a gel
August 18, 2009
- Tranfected ligation products into DH-5alpha cells and plated on old plates
- Miniprepped overnight culture of 7
- Digested Enc6, C (E,P)
- ran a gel with digests
- Digested Enc 6, C (X,S)
- Ran a gel with digests
- Gel extracted C (CIPed)
- Started overnight cultures for Tet, K plasmid
- Started overnight digest for Enc6
August 19, 2009
- Miniprepped overnight cultures of Tet, K
- Ran a gel of the ligations and the overnight digest
- Started overnight culture of 4+5 colonies
- Ligated Enc into CIPed C backbone
- Started overnight K and Tet digestion
- Poured Tet plates
August 20, 2009
- Digested C plasmid
- Ran gel of C, K, Tet digests
- Miniprepped 4+5 overnight cultures -> 4+5?
- Started overnight cultures of Tet and a negative control
August 21, 2009
- No growth of negative Tet overnight culture
- Tet overnights were miniprepped
- Digested 4+5?, Tet, and TetX with (EcoR1 and Pst1)
- The digests were run on a gel with negative controls
- None of the samples were confirmed
- Started overnights for Tet plasmid, 2 with tetracyclin and 1 with ampicillin
August 22, 2009
- 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
- Tet1: Did 2 minipreps, one with normal protocol, one with low copy plasmid protocol
- Digested the 2 samples with higher yields (500ng)
- Ran digests on gel, but still couldn't see ccdb gene
- Started to doubt restriction enzyme function, therefore redigested Tet H (1ug) and C plasmid (500ng)
- (1.5h digests for all digests today)
- 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
- Started overnight ligation of 4+5 in Tet backbone
August 23, 2009
- Transfected 4+5, Enc+C (x2) ligations into 1 tube of Calvin's cells (aliquots of 33ul of cells)
- plated Enc transformants onto C plates
- Since there were no Tet plates, 400ul of tetracyclin was spread onto a plain LB plate and dried, before plating 4+5 transformants
- Started overnight cultures of 1+2 and 3+2 in 5ml of LB
August 24, 2009
- Replated left over cells
- Miniprepped 1+2 overnights, yielded ~40ng/ul
- 3+2(2) showed no growth
- Plates in the incubator showed no colonies
August 25, 2009
- Enc replate showed 3 colonies
- Started a log phase growth of 3+2(2), but it showed not growth again
- 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
- 4+5 plates showed 2 big colonies and 1 small colony
- Started log phase for 4+5 (1), (2) (did not stock)
- 3+2 overnights did not grow
- They are not C-resistant?
- We have to redo the 3+2 ligations
- Miniprepped Enc A, B, C, BB3 and BB4
- Digested miniprepped samples and also BB2, 1+2 plasmid for 2 hours
- 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)
- 30x37.4=1122ng of plasmid was digested for 1+2; and 16.5ul was loaded (1122x16.5/50=370.26ng)
- Miniprepped log phase of 4+5 (1), (2)
August 27, 2009
- Digested 4+5 (1), (2), BB7 with E+P
- Ran digstes on gel
- wall between lane 5 and 6 was broken upon loading
- religated BB3 digest and 3+2 into C plasmid for 2 hours at room temperature
- plated ligations: 2ul of ligation in 25ul of cells
August 28, 2009
- Transfected 50ul of DH5 cells with 4+5(1) plasmid (2ul)
- added 100ul of LB+glucose and plated all on a Tet plate
- Mixed up LB+agar for pouring C plates on Monday (Aug 31)
- Picked colonies growing on 4+5 plate and Enc plate and started overnight cultures
August 29, 2009
- Stocked Enc, 4+5 overnights and stored remaining culture in the fridge (4C)
- 4+5 retransfection plate did not show colonies
August 30, 2009
- 4+5 retransfection plate had full plate of colonies
- Overnight cultures of 4+5 and BB7 were started
August 31, 2009
- Stocked 4+5R culture (retransfection)
- Miniprepped overnight cultures of 4+5 (3,4,5,R), BB7, and EncX,Y,Z
- Digested EncC with E,S; 5 with X,P; Tet, 7, EncX,Y,Z with E,P
- 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
- Poured C plates
- 3+2 tranformation from Aug 27 showed a few colonies
- 3 largest colonies were picked to start overnight culture
- Started overnight ligation of Enc+5/Tet with negative control