Team:TorontoMaRSDiscovery/Notebook

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#*incubated on shaker for about 2h
#*incubated on shaker for about 2h
#Started overnight of C plasmid (ccdb)
#Started overnight of C plasmid (ccdb)
 +
 +
=September 5, 2009=
 +
#Ran a gel of digests from yesterday with controls
 +
#Miniprepped C overnight culture
 +
#Tet plate (Tet with RFP) showed 2 red colonies
 +
 +
=September 7, 2009=
 +
#Digested C plasmid and redigested Enc+5 (1), (3), K1, K2
 +
#Started Tet1, Tet2 overnight culture
 +
 +
=September 8, 2009=
 +
#Ran a gel of the digests
 +
#*Enc+5 band still not showing may have to religate
 +
#*K and C plasmids digests showed inserts
 +
#Started log phase culture for Tet1, Tet2
 +
#1+2+3+2 plate still hasn't shown any colonies#Ran digests on a gel
 +
#Started overnight ligation of 1+2+Enc, 1+2+3+2 in K1
 +
 +
=September 9, 2009=
 +
#Transfected cells with overnight ligations
 +
#*2 samples per ligation
 +
#*Plated and grew overnight at 37C
 +
 +
=September 10, 2009=
 +
#Saw lots of growth on plates (no red colonies yet)
 +
#Prepared 10 overnight growth placed in incubator at 30C in K antibiotics
 +
#put plates back in incubator at 37C
 +
 +
=September 11, 2009=
 +
#Overnight cultures (x5) were miniprepped
 +
 +
=September 12, 2009=
 +
#Started 20h digest of miniprepped samples
 +
 +
=September 13, 2009=
 +
#Ran digests and negative controls on gel, but no bands at all were seen.
 +
 +
=September 14, 2009=
 +
#Started overnight cultures of same 5 samples
 +
 +
=September 15, 2009=
 +
#only 1232 (3) showed growth, miniprepped
 +
#started overnight cultures of for 1232 (2), (4), 12E (2) and EncY
 +
 +
=September 16, 2009=
 +
#Only EncY showed growth, miniprepped
 +
#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=
 +
#1232 mix1, 2 and 12E mix2 showed growth
 +
#PCRed EncY plasmid and ran on gel
 +
#*a huge ~700bp band was seen
 +
#Prepared EncY for sequencing
 +
 +
=September 18, 2009=
 +
#Digested 1232 (3), 1+2, 7, EncY, K
 +
#Ran digests on gel
 +
#*7 did not show up on gel
 +
#*Enc band was faint/did not show up
 +
 +
=September 19, 2009=
 +
#12E replates showed some colonies -> started overnights: 12E a,b,c,d,e
 +
#started overnight culture for EncY
 +
#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=
 +
#Miniprepped overnights except 12E (which did not show any growth in LB with antibiotics)
 +
#Digested 7,K, EncY, 12E a,b,d,e
 +
#Ran digests on gel
 +
#*only Enc was confirmed on this gel
 +
#Started K1 overnight
 +
 +
=September 24, 2009=
 +
#Made a new batch of chemically competent cells
 +
#*poured cells into 50ml falcon tubes
 +
#*350ul aliquots + 350ul 40% glycerol
 +
#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
 +
#Transformed ligation from Monday and negative control into new cells and plated on new K plates
 +
 +
=September 25, 2009=
 +
#Miniprepped 12E 6-10, f-j
 +
#*overnights from 9,10,g,i had almost no growth (confirmed after 1st centriugation step: did not miniprep)
 +
#Digested miniprepes and ran on gel -> no bands
 +
#Started overnights again
 +
 +
=September 26, 2009=
 +
#Only 12E (6),f,j,h
 +
#Digested minipreps
 +
#Ran a gel
 +
 +
=September 28, 2009=
 +
#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=
 +
#took 400ul aliquots of the 4 samples for storage as stocks
 +
#Miniprepped the rest of the samples
 +
#Ran gel saw no colonies
 +
#Rechecked plates: lack of red colonies is suspicious
 +
#*DH5 may be somewhat resistant to K (suggested by Calvin)
 +
#May need to gel extract backbone and parts before ligation
 +
#We are out of K plates; need to make more
 +
 +
=October 1, 2009=
 +
#Miniprepped Tet, 5, EncY, C
 +
#Ran minipreps and K plasmid on a gel
 +
#*Besides Tet, all samples looked fine
 +
#Poured K plates
 +
 +
=October 2, 2009=
 +
#Digested K, EncY and ran on gel
 +
 +
=October 3, 2009=
 +
#Digested EncY and old Tet plasmid -> ran on gel
 +
#*EncY looked fine, but Tet did not show up on gel
 +
#Started overnight culture of Tet
 +
 +
=October 4, 2009=
 +
#Miniprepped, digested Tet and ran on gel
 +
#*Tet was confirmed
 +
#gel extracted EncY, Tet and got low yields
 +
#Started overnight ligations
 +
#*1: Digested Tet + EncY + 5
 +
#*2: Gel extraced Tet + Digested EncY + 5
 +
#*negative controls were Tet plasmid alone
 +
 +
=October 8, 2009=
 +
 +
=October 9, 2009=
 +
#Miniprepped EncY overnight and digested with E,P
 +
#Ran digest on a gel
 +
#*Enc part confirmed
 +
#Transfected Enc+5 ligations from Sunday/Monday into competent cells from Invitrogen
 +
#*Used NEB Hight Efficiency Transformation Protocol C2987
 +
#**did one 10-fold dilution
 +
#**transformed 5ul of DNA
 +
#**spread 100ul of cells on plate
 +
#**shaker/incubator speed 232rpm
 +
 +
=October 11, 2009=
 +
#Checked plates, no colonies yet
 +
#Picked colonies from an old 12E plate that wasn't checked: 12E i,ii,iii
 +
 +
=October 13, 2009=
 +
#12E i and iii showed growth -> miniprepped
 +
#Digested and ran on 1% agarose gel
 +
#*Stained gel for 1 hour in EtBr+buffer
 +
#*A faint ~700 band was observed! This could possibly be the 1+2+Enc insert that we want.
 +
#To confirm the insert, we decided to sequence it
 +
 +
=October 15, 2009=
 +
#Primers for sequencing 12E insert was designed and ordered
 +
 +
=October 16, 2009=
 +
#EncY prepped and ready to ship down to MIT to submit part to registry

Revision as of 21:25, 18 October 2009

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Home The Team The Project Parts Submitted to the Registry Modeling Notebook


Contents

April 27, 2009

  1. Received from Rosa (SPiT):
    • TM0785
      • Plasmid containing encapsulin
      • Recommend transfect into bacteria and re-sequence
      • See email note regarding sequence error
    • 0.5 microliters TMG DNA 100 microgram/microliter
      • Use 0.4 microliter for 50 microliter PCR reaction =August 1, 2009=
  2. Overnight encapsulin cultures 1 and 6 (randomly selected) and the negative control were miniprepped
  3. The rest of the encapsulin cultures were stocked with 20% glycerol
  4. 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
  5. The digestions were run on a 1.3% agarose gel in TAE (from here on, unless otherwise specified, all gels were 1.3% agarose)
    • BB5 was confirmed and all other parts were correct as well
  6. 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

May 15, 2009

  1. pH buffers received from VWR Mississauga
    • pH 4 buffer (red) 500 ml
    • pH 7 buffer (yellow) 500 ml
    • pH 10 buffer (blue) 500 ml
      Above are used for pH/mV Meter calibration
  2. pH/mV meter calibrated according to manual – recorded in index
  3. Ethanol solution (70%) made from 85% ethanol

May 19, 2009

  1. 2L of TE buffer made (10X TE)
    Recipe for 2L from stock solution (10X TE)
    a) 10 ml 1M Tris-HCl pH 7.5-80.0 x 2 = 20 ml
    b) 2 ml 0.5 M EDTA pH 8.0 x 2 = 4 ml
    c) 988 ml ddH20 x 2 = 1976 ml
    To make Tris-HCl, Tris base is used and adjusted to desired pH using HCl
    1 M Tris base is required of which 20 ml is required – thereby 2.4228 g Tris base (due to scale limitations, 2.42g of Tris Base was used
  2. 500 ml of 1 M Tris Base made
    mass of Tris used = 1M/1000 ml x 500 ml x 121.14 g/mol = 60.57 g
    volume of water used = 500 ml
  3. 250 ml of 0.5 M EDTA solution was made
    mass of EDTA used = 36.53 g
    observations: EDTA did not dissolve in ddH2O on heat and being stirred

May 21, 2009

  1. Retrieved autoclaved ddH20, glycerol solution
  2. Gel Electrophoresis (test run)
    • 1% gel: 0.5 g agar mixed with 50 ml of 1X TBE
    • 10X TBE diluted with 5 ml TBE + 45 ml ddH20 – did not use all 50 ml for 1 gel (gel will be too thick – make 2 instead)
    • Loading Dye: add glycerol solution to 25 mg bromophenol blue then add dH20 to 10 ml (to make 6X)
    • Running gel: match wells to black side, run at 120 mA
  3. Visualize Gel in UV
    1. Turn power on
    2. Gel in machine face up
    3. Close door securely
    4. Turn white light on
    5. Adjust zoom, contrast, focus from black dial on top of machine
    6. Turn white light off (turns on UV)
    7. Press ‘live’ toggle – acq. Should be 0.4 sec.
    8. Print if desired or save on floppy disk
    9. Turn power off
    10. Dispose of gel in proper container
    11. Close door

May 25, 2009

  1. Made overnight bacteria culture (200 ml LB: 20 microliters DB3.1)

May 26, 2009

  1. Took overnight cultures from incubator
  2. Inoculated 2 500 ml flasks with 25 ml of overnight culture (1 flask/culture)
  3. Placed 500 ml flasks into incubator at 37 degrees Celcius
  4. Grew overnights of DB3.1 from Waterloo (thanks :))

June 3, 2009

  1. Plasmid transformed = pSB1AC3 (TEST)
    • Note: team members were of mixed opinion as to whether the transform was likely due to low availability of DNA - DNA was split between a number of transformation reactions for at least 2 different DB3.1 strains. Plates incubated at 30 degrees Celsius overnight, temp. raised to 37 degrees approx. 10:30 a.m.

June 5, 2009

  1. Tet plates made
    Recipe for 200 ml (approx. 10 plates):
    2.2 g agar in 200 ml fresh LB
    Note: do not re-autoclave LB, it will caramelize!
    Recipe for 200 ml LB:
    a) 1 g yeast extract
    b) 2 g peptotryptone
    c) 2 g NaCl
    d) 200 ml water
  2. Autoclaved at 11:20 a.m., picked up at 1:15 p.m. and cooled in water bath at 37 degrees Celsius
  3. Added 200 microlitres tetracycline stock (from -20 freezer) to final concentration of 20 microgram/ml
  4. Swirl and poured into prepared, labeled plates
    • Note: label on plates reads June 3, 2009 but should be June 5, 2009 (8 plates in total - either I overpoured or these plates are a bit larger than I was used to)
  5. Inverted and put in 37 degree incubator to dry

June 8, 2009

  1. Bacteria grown to log phase using sterile inoculating loop to inoculate approx. 200 ml of LB media with bacteria from 2 plates (approx. 3 colonies)
  2. Bacterial liquid culture placed in shaker at 10:51 a.m.

June 9, 2009

  1. Digested miniprepped gel with EcoRI and SpeI
  2. Running gel to see if correct plasmid was purified - should expect to see bands at ~3200 bp
  3. DNA Ladder made - 6 microlitres of stock used

June 10, 2009

  1. Poured 10 Tet plates following procedure on June 5, 2009
  2. Due to problems with DNA ladder and restriction enzyme digest yesterday, procedures were followed again today with the following adjustments
    • DNA was diluted and run on lanes 1-5 of gel:
      • Lane 1 - 1X
      • Lane 2 - 1/6X
      • Lane 3 - 1/36X
      • Lane 4 - 1/10X
      • Lane 5 - 1/100X
    • Restriction enzyme digest was repeated with an incubation time of 4 h at 37 degrees Celsius and heat shock at 80 degrees Celsius was done in hot water bath
    • Results: Dilution did not work well (ladder could not be visualized). Digest appeared to work as 3 bands could be seen in last 2 lanes
    • Adjustments for tomorrow:
      • Spin down enzymes before using
      • Overnight digest

June 11, 2009

  • We did not have enough isolated plasmid DNA to justify doing another gel so we decided to perform another gel so we decided to perform another miniprep to get some more
  • Used DB3.1 transformed bacteria from shaker(measured spec 0.794)
  • Miniprep
    • potential issue our microcentrifuge only spins @ 10,000 not required 12,000
  • Binding DNA
    • did both optional steps preheated TE + washed w/ W10
  • measured UV absorbance = 4.2ng/ul
  • in all there is ~ 74 ul of TE should indicated ~370 ng of plasmid.
  • Digest
    • NOTE: plasmid length w/e biobric is 3189 bp biobrick length is 3255bp this can explain lack of 2 clear bands on gel after ligation.
    • nevertheless, we will perform another Gel to make sure we can properly run ladder to determine fragment sizes
  • digest measurements: 250ng plasmid all else half except BSA

June 12, 2009

  • Ran Gel
    • Ladder and other bands were able to be visualized however were still a little wonky
    • Suggests for improvement
      • increase the glycerol [] in loading dye as there have been problems with diffusion when loading samples as the solution is not falling into the wells

June 15, 2009

  • made 1ml and 200ul aliquotes of Kanamycin 10mg/ml stock and stored at -20C
    • Recipe: 250ug Kanamycin + 25ml sterile water
    • USE : 2x200ul for final working concentration of 20ug/ml

Trouble shooting ladder

  • it was noted that the 1x TBE buffer recipe was off. The

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

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

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#Ran digests on a gel
  4. 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

October 1, 2009

  1. Miniprepped Tet, 5, EncY, C
  2. Ran minipreps and K plasmid on a gel
    • Besides Tet, all samples looked fine
  3. Poured K plates

October 2, 2009

  1. Digested K, EncY and ran on gel

October 3, 2009

  1. Digested EncY and old Tet plasmid -> ran on gel
    • EncY looked fine, but Tet did not show up on gel
  2. Started overnight culture of Tet

October 4, 2009

  1. Miniprepped, digested Tet and ran on gel
    • Tet was confirmed
  2. gel extracted EncY, Tet and got low yields
  3. Started overnight ligations
    • 1: Digested Tet + EncY + 5
    • 2: Gel extraced Tet + Digested EncY + 5
    • negative controls were Tet plasmid alone

October 8, 2009

October 9, 2009

  1. Miniprepped EncY overnight and digested with E,P
  2. Ran digest on a gel
    • Enc part confirmed
  3. Transfected Enc+5 ligations from Sunday/Monday into competent cells from Invitrogen
    • Used NEB Hight Efficiency Transformation Protocol C2987
      • did one 10-fold dilution
      • transformed 5ul of DNA
      • spread 100ul of cells on plate
      • shaker/incubator speed 232rpm

October 11, 2009

  1. Checked plates, no colonies yet
  2. Picked colonies from an old 12E plate that wasn't checked: 12E i,ii,iii

October 13, 2009

  1. 12E i and iii showed growth -> miniprepped
  2. Digested and ran on 1% agarose gel
    • Stained gel for 1 hour in EtBr+buffer
    • A faint ~700 band was observed! This could possibly be the 1+2+Enc insert that we want.
  3. To confirm the insert, we decided to sequence it

October 15, 2009

  1. Primers for sequencing 12E insert was designed and ordered

October 16, 2009

  1. EncY prepped and ready to ship down to MIT to submit part to registry
Retrieved from "http://2009.igem.org/Team:TorontoMaRSDiscovery/Notebook"