Team:TorontoMaRSDiscovery/Notebook/Augst



=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
 * 4) *16ul of BB5 plasmid was used
 * 5) *500ng of plasmid were used for the others
 * 6) The digestions were run on a 1.3% agarose gel in TAE
 * 7) *BB5 was confirmed and all other parts were correct as well
 * 8) 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)
 * 3) *The 3kbp in the digest of 1+2 Sample 5 was not expected
 * 4) *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.
 * 5) 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
 * 3) *All bands were of expected sizes.
 * 4) 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
 * 3) *The 1+2 insert was not seen on the gel, probably because it is too small
 * 4) *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
 * 4) **This is thumotoga maritime genomic DNA for purpose of re-cloning 
 * 5) Microcentrifuge tubes 1 and 2 placed in -20 freezer

=August 8, 2009=
 * 1) BB1 transformants showed many colonies
 * 2) *plates (a plate full of colonies)
 * 3) *-> increasing heat shock to 90s increased efficiency (Note: 3x DNA used in this transformation)
 * 4) *Calvin mentioned he does heat shock for 120s
 * 5) *Calvin also suggested to increase tranformation time, ie. after adding DNA, hold on ice for an hour
 * 6) DH-5alpha replates all grew on the plates, demonstrating LB can be re-autoclaved and poured as plates
 * 7) *there was no difference between transformants directly plated after adding LB+glucose or incubating for an hour
 * 8) 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
 * 3) *(1+2) Sample 1 appeared to have a ~100bp band
 * 4) *K plasmid digest did not show the ccdb gene (~700bp) - will start another culture with antibiotics
 * 5) Started overnight cultures of (1+2) Sample 1

=August 11, 2009=
 * 1) Digested 4, 5, Enc, C(E+P), C(X+S)
 * 2) *BB4 did not digest
 * 5, C is stored in fridge
 * 1) Calvin suggested increasing the concentration of enzyme and increase time of digestion due to thawing enzymes over weekend in fridge
 * 2) Miniprepped 1+2(1) with new kit, got great yield ~100ng/ul
 * 3) Gel extracted C (X+S) after it was CIPed
 * 4) Overnight ligation of Enc+C in PCR machine
 * 5) Calvin gave us 2 vials of RbCl cells in -80C
 * 6) *one time use only
 * 7) *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
 * 6) *not enough agar?

=August 14, 2009=
 * 1) Miniprepped 5, 7, C with normal protocol of BioBasics Kit
 * 2) *Results were low ~30ng/ul
 * 3) Digested C and CIPed it
 * 4) 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
 * 5) Gel extracted CIPed C plasmid
 * 6) 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
 * 2) *7 sample had a very low yield
 * 3) *started another overnight for & and will miniprep again tomorrow
 * 4) Digested (1+2) Sample 1, (3+2) Sample 2, 4, 5 and K plasmid
 * 5) Started an overnight ligation for (1+2)+(3+2) and 4+5 in K plasmid
 * 6) 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
 * 5) *None of the samples were confirmed
 * 6) Started overnights for Tet plasmid, 2 with tetracyclin and 1 with ampicillin

=August 22, 2009=
 * 1) Miniprepped overnights:
 * 2) *Tet1: Did 2 minipreps, one with normal protocol, one with low copy plasmid protocol
 * 3) **Normal protocol gave a higher yield
 * 4) *Tet with ampicillin: used low copy plasmid protocol, but cell pellet was much bigger therefore got the same yield as Tet1 normal protocol
 * 5) Digested the 2 samples with higher yields (500ng)
 * 6) Ran digests on gel, but still couldn't see ccdb gene
 * 7) Started to doubt restriction enzyme function, therefore redigested Tet H (1ug) and C plasmid (500ng)
 * 8) (1.5h digests for all digests today)
 * 9) Ran Tet H and C digests on a gel
 * 10) *A faint ~700bp band was observed in the Tet lane
 * 11) *More plasmid should be used for plasmid digests
 * 12) 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:
 * 4) *Enc A, B, C
 * 5) *3+2 (2), (3), (4), (5), and a 3+2 (2) positive control (in LB only)
 * 6) *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
 * 4) *They are not C-resistant?
 * 5) *We have to redo the 3+2 ligations
 * 6) Miniprepped Enc A, B, C, BB3 and BB4
 * 7) Digested miniprepped samples and also BB2, 1+2 plasmid for 2 hours
 * 8) Ran digests on a gel
 * 9) *1ug of plasmid was digested for Enc A, B, C, BB2, BB3, BB4; and 1/5 was loaded on to gel (10ul of digest)
 * 10) 30x37.4=1122ng of plasmid was digested for 1+2; and 16.5ul was loaded (1122x16.5/50=370.26ng)
 * 11) 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
 * 3) *wall between lane 5 and 6 was broken upon loading
 * 4) religated BB3 digest and 3+2 into C plasmid for 2 hours at room temperature
 * 5) 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
 * 5) *EncY shows Enc insert
 * 6) *5 digest lane shows a thick band the same size of the linear isoform in the control
 * 7) Poured C plates
 * 8) 3+2 tranformation from Aug 27 showed a few colonies
 * 9) 3 largest colonies were picked to start overnight culture
 * 10) Started overnight ligation of Enc+5/Tet with negative control