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| Line 10: |
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| | | | |
| - | =April 27, 2009= | + | =April= |
| - | #Received from Rosa (SPiT):
| + | =May= |
| - | #*TM0785
| + | =June= |
| - | #**Plasmid containing encapsulin
| + | =July= |
| - | #**Recommend transfect into bacteria and re-sequence
| + | =August= |
| - | #**See email note regarding sequence error
| + | =September= |
| - | #*0.5 microliters TMG DNA 100 microgram/microliter
| + | =October= |
| - | #**Use 0.4 microliter for 50 microliter PCR reaction =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 (from here on, unless otherwise specified, all gels were 1.3% agarose)
| + | |
| - | #*BB5 was confirmed and all other parts were correct as well
| + | |
| - | #Overnight ligation of 7+Enc in the PCR machine
| + | |
| - | | + | |
| - | =May 15, 2009= | + | |
| - | #pH buffers received from VWR Mississauga
| + | |
| - | #*pH 4 buffer (red) 500 ml
| + | |
| - | #*pH 7 buffer (yellow) 500 ml
| + | |
| - | #*pH 10 buffer (blue) 500 ml<Br />''Above are used for pH/mV Meter calibration''
| + | |
| - | #pH/mV meter calibrated according to manual – recorded in index
| + | |
| - | #Ethanol solution (70%) made from 85% ethanol
| + | |
| - | | + | |
| - | =May 19, 2009=
| + | |
| - | #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'' <Br />
| + | |
| - | #::''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''
| + | |
| - | #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
| + | |
| - | #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=
| + | |
| - | #Retrieved autoclaved ddH20, glycerol solution
| + | |
| - | #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
| + | |
| - | #Visualize Gel in UV
| + | |
| - | ##Turn power on
| + | |
| - | ##Gel in machine face up
| + | |
| - | ##Close door securely
| + | |
| - | ##Turn white light on
| + | |
| - | ##Adjust zoom, contrast, focus from black dial on top of machine
| + | |
| - | ##Turn white light off (turns on UV)
| + | |
| - | ##Press ‘live’ toggle – acq. Should be 0.4 sec.
| + | |
| - | ##Print if desired or save on floppy disk
| + | |
| - | ##Turn power off
| + | |
| - | ##Dispose of gel in proper container
| + | |
| - | ##Close door
| + | |
| - | | + | |
| - | =May 25, 2009=
| + | |
| - | #Made overnight bacteria culture (200 ml LB: 20 microliters DB3.1)
| + | |
| - | | + | |
| - | =May 26, 2009=
| + | |
| - | #Took overnight cultures from incubator
| + | |
| - | #Inoculated 2 500 ml flasks with 25 ml of overnight culture (1 flask/culture)
| + | |
| - | #Placed 500 ml flasks into incubator at 37 degrees Celcius
| + | |
| - | #Grew overnights of DB3.1 from Waterloo (thanks :))
| + | |
| - | | + | |
| - | =June 3, 2009= | + | |
| - | #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=
| + | |
| - | #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
| + | |
| - | #Autoclaved at 11:20 a.m., picked up at 1:15 p.m. and cooled in water bath at 37 degrees Celsius
| + | |
| - | #Added 200 microlitres tetracycline stock (from -20 freezer) to final concentration of 20 microgram/ml
| + | |
| - | #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)
| + | |
| - | #Inverted and put in 37 degree incubator to dry
| + | |
| - | | + | |
| - | =June 8, 2009=
| + | |
| - | #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)
| + | |
| - | #Bacterial liquid culture placed in shaker at 10:51 a.m.
| + | |
| - | | + | |
| - | =June 9, 2009=
| + | |
| - | #Digested miniprepped gel with EcoRI and SpeI
| + | |
| - | #Running gel to see if correct plasmid was purified - should expect to see bands at ~3200 bp
| + | |
| - | #DNA Ladder made - 6 microlitres of stock used
| + | |
| - | | + | |
| - | =June 10, 2009=
| + | |
| - | #Poured 10 Tet plates following procedure on June 5, 2009
| + | |
| - | #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= | + | |
| - | #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 <Br />
| + | |
| - | #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
| + | |
| - | #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
| + | |
| - | | + | |
| - | =September 1, 2009= | + | |
| - | #only 3+2a overnight showed growth
| + | |
| - | #Miniprepped 3+2a
| + | |
| - | #digested 3+2a, 4+5R, 3, 4, 5 with E,P
| + | |
| - | #Transfected EncC+5 and negative control into DH-5 cells, plated on Tet plates
| + | |
| - | #mistakenly tranfected T, K plasmid into DH-5 cells (ccdb gene)
| + | |
| - | #Ran digests on a gel
| + | |
| - | #*3+2 still not confirmed, but all 4+5 samples showed expected band
| + | |
| - | | + | |
| - | =September 2, 2009=
| + | |
| - | #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
| + | |
| - | #Enc+5 showed larger colony -> started overnight
| + | |
| - | | + | |
| - | =September 3, 2009=
| + | |
| - | #only 1+2 (1), (4) showed growth in C
| + | |
| - | #Miniprepped Enc+5, 1+2 (1), (4)
| + | |
| - | #Digested Enc+5 with E,P, 1+2 (1), (4) with E,S, 3+2a with X,P for 1.5h
| + | |
| - | #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
| + | |
| - | #Started overnight ligation of 1+2+3+2/Tet
| + | |
| - | #Started overnight cultures: Ampx1, Kx2 Enc+5 x3, Tet, BB7
| + | |
| - | | + | |
| - | =September 4, 2009=
| + | |
| - | #Miniprepped overnight cultures
| + | |
| - | #stocked Amp, K1, K2, and Enc+5 (2), (3), (4)
| + | |
| - | #Digested Enc+5 (1)-(4), K1,2, BB7
| + | |
| - | #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
| + | |
| - | #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 and started overnights 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
| + | |
"
