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May 2012

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  • SU | MO | TU | WE | TH | FR | SA

June 2012

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  • SU | MO | TU | WE | TH | FR | SA

July 2012

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August 2012

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September 2012

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  May 17

  • 16 plates each of LB agar, ampicillin, tetracycline, and chloramphenicol were made.
  • To test the effectiveness of our antibiotic stock, five vials were created, and were kept in the shaker.
  • Vial one contained LB control; vial two contained LB and DH5α; vial three contained LB, DH5α, and ampicillin; vial four contained LB, DH5α, and chloramphenicol; vial five contained LB, DH5α, and tetracycline.

 May 18

  • Placed all the agar plates in the fridge, as no contamination was visible. Vial two was cloudy, as expected, indicating DH5α growth. The large block of ice formed over ten years' neglect in the 4°C fridge below our lab bench was cleaned with a chisel and mallet.
  • Four plates were prepared with DH5α: LB agar; LB ampicillin plate; LB tetracycline plate; LB chloramphenicol plate.
  • Vial Two was disposed of after bleaching. Stocks of 70% and 100% EtOH were obtained free from the Bendeck lab upstairs, replenishing our meagre stock.

  May 22

  • Made 1L of LB, as well as autoclaved some glassware. Only plate one from May 18 showed any bacterial growth after incubation at 4°C overnight, indicating good sterile technique during the training exercises yesterday.
  • SNC Lavalin, Monsanto, and New England BioLabs were contacted for funding enquiries. Approached Rob Reedijk about consolidation of financial accounts. Obtained A. thaliana seed from the McCourt lab at EEB, along with their recommended growth protocol.

  May 23

  • For demonstration purposes, we transformed cyan fluorescent protein from the BioBrick distribution plates with ampicillin resistance into DH5αC (competent DH5α).

  May 24

  • Checked the cyan fluorescent transformation, which failed. To check whether the BioBrick plasmids were compromised, or whether our technique was to blame, we will retry the transformation with another BioBrick. Ordered supplies from MedStore.

  May 25

  • Made kanamycin plates, and checked on the May 24 transformations, which proved to be successful. Aimed to finalise the phytase construct.

  May 28

  • Various sponsorship opportunities were pursued for the lab for much of the day.
  • 2 test transformations were performed as another tutorial. Kanamycin's efficiency was tested with the following three treatments: LB; LB and DH5αC; LB, DH5αC, and K.

  May 29

  • Tested a 2µL/mL kanamycin overnight culture versus an LB blank on the spectrophotometer. Placed two different projects into the shaker: Project One (10mL LB; 10µL C; 20µL DH5α): LB control; LB, DH5α, and C; LB, DH5α, C, and BioBrick K414007. Project Two (10mL LB; 20µL DH5α): LB control; LB and DH5α; LB, DH5α, and 20µL K; LB, DH5α, and 25µL K; LB, DH5α, and 30µL K.

  May 30

  • The LB controls failed from yesterday, so the 500mL container of LB from which we took yesterday's samples was thrown out. The K414007 promoter was miniprepped using a Qiagen Plasmid Extraction Kit. Using the nanodrop, we found our extraction sample to have a density of 20ng/µL – good miniprep. Performed a transformation using K414007, so as to keep plates in the fridge with this part handy.

  June 1

  • Competent E. coli were made using the CaCl2 protocol. The transformation from yesterday was successful. We put K414007 into the -20°C freezer. The primers for the following BioBricks we intend to make as part of our constructs were designed: Kozak, HSP18.2 terminator, PHT1;2 promoter, Extensin signal, AtPAP15, and PhyA1 (which has two exons, and therefore, four primers needed). We also miniprepped the K382024 terminator, and made a long-term stock as well as a lab stock.

  June 4

  • The BioBrick K414007 was grown up so that it was possible to make a long-term stock. We did so by inoculating a colony on an LB and chloramphenicol plate. Our competent cells from June 1 were tested by transforming K414007. A restriction digest was run on K382024 using EcoR1 (E) and Spe1 (S).

  June 5

  • K414007 was miniprepped and a long-term stock was made by suspension in 1000µL 40% glycerol and placed in the -80°C. The nanodrop showed densities for K414007 of 78 and 100 ng/µL. As a test for competency, the newly-made competent DH5α was transformed successfully. We now have a stock of 49 aliquots in the -80°C. The chemical cupboard in the lab was also organised.

  June 7

  • A 20% SDS solution was made for DNA isolation next week, and PCR primer designs were finalised.

  June 8

  • The PCR primers were ordered – desalted, 25 nmole, with no 5' or 3' modifications. The following four strains of Aspergillus from Sporometrics: (i) SCCM# 11-B5 A. niger (Sporometrics Internal QC); (ii) SCCM# 15-E9 A. brasiliensis (Environmentally acquired); (iii) SCCM# 17-B6 A. niger (Environmentally acquired); and (iv) SCCM# 11-B6 A. niger (Sporometrics Internal QC).
  • Made LB agar, into which we added kanamycin from old stock we found in the chemical storage cupboard and was more effective than the stock we bought last month. (Remember that the antibiotic solution is added to the LB agar after it is autoclaved, but before it has cooled to solidification) Two plates of each Aspergillus strain were prepared in PD agar in order to make a long-term stock. The plates were left to culture over the weekend at 37°C. MS medium was made, and A. thaliana seeds were inoculated on the plates, which were stored in the cold room at 4°C.

  June 11

  • Made a long-term stock of all Aspergillus strains by suspending in 40% glycerol and storing in the -80°C. Attempted a Chelex prep on the A. niger strain 11-B5, which failed. Transformed K652001 using DH5alphaC.

  June 12

  • Made 500mL CTAB extraction buffer for D. carota DNA extraction, and 500mL TENS buffer for A. niger DNA extraction. The fume hood was cleaned, and all the chemicals were organised therein. The K652001 transformation was done with two plasmids (since the BioBrick kit came with two plasmids): one failed, the other did not. As a result, we must send this DNA for sequencing before continuing with PCR on this BioBrick. The D. carota DNA extraction was attempted, but had to be postponed, because the proteinase K stock exploded in the -20°C.

  June 13

  • Digested and ligated the Kozak sequence and did a transformation. The D. carota DNA extraction was attempted, and failed because regular 15mL Falcon tubes containing the DNA samples broke in the refrigerated centrifuge.

  June 14

  • The first portion of the D. carota DNA extraction was completed, and the result was left to incubate in the -20°C overnight, as per the extraction protocol. The June 13 Kozak transformations failed, so another attempt shall be made to remedy this situation.

  June 15

  • UTSU was contacted in order to begin the process of cheque re-issue for monies owed to iGEM. The lab bench was re-organised in order to make everything easier to access. The D. carota genomic DNA extraction was completed. A grow-box was made with red and blue LEDs such that the A. thaliana could grow properly under regulated lab conditions. The following solutions were made in the lab: 1L 5M NaCl, 500mL 20% SDS, 500mL 3M NaOAc pH 5.3, 500mL 25mM MgCl2, and 500mL 7.5M NH4OAc.

  June 18

  • Another ligation and transformation of the Kozak sequence was attempted. We tried making an agarose gel, but it did not solidify in time, so the gel was postponed until June 19. The 1% agarose gel was to run the carrot genomic DNA. The two carrot gDNA samples were nanodropped: sample one (1) has a density of 68.4 ng/µL, and sample two (2) has a density of 91.4 ng/µL.

  June 19

  • Got $300 contribution from the Skule Alumni Fund. Went on an outing to Queen St. W. to get an amber bottle, electrical wiring, and a scalpel. Made a 1% agarose gel, which broke (again!) and made 400mL of 24:1 chloroform: isoamyl alcohol, as well as 1L Tris-HCl, pH 8.0.

  June 20

  • Made 1L each of Super Optical Both with Catabolite repression (SOC) and Terrific Broth. Made a 1% agarose gel, which broke...again. Made yet another gel, using 1.2g agarose, 120mL 1x TBE buffer, and a voltage of 60V. This procedure worked, and the carrot DNA samples were run, and DNA presence in the samples was confirmed. The gels were accidentally thrown out after imaging. A gDNA prep of the A. niger was also done, with some modifications from the original protocol. Some A. thaliana were planted in pots from the MS-K plates they were cultured on. They were excised by scalpel and transferred to pots filled with potting soil to an approximate 4" height. The plants were watered with milli-Q.

  June 21

  • The shaker was observed to see if its malfunction one week ago was purely by chance; it appeared to be so, and thus, the following five treatments were placed into the shaker for overnight incubation: (i) LB control; (ii) LB, DH5alpha, ampicillin; (iii) LB, DH5alpha, chloramphenicol; (iv) LB chloramphenicol and K652001; and (v) LB ampicillin, and psb1A3.
  • A 1% agarose gel (made using the 1.2g agarose recipe discussed previously) was made, and the A. niger DNA prep was run. The gel was inconclusive, and must be run again tomorrow. The DNA primer melting temperatures were determined and posted for the team on Dropbox. A 100x Antibiotic-antimycotic solution, composed of amphotericin B, streptomycin, and penicillin was bought for use on the potted plants. The solution was aliquoted into 15mL Sarstedt tubes, placed in the -20°C. A few tubes were thawed, diluted ten-fold, and transferred to a spray bottle. The solution will be sprayed on twice weekly henceforth.

  June 22

  • The K652001 and psb1A3 treatments from the shaker were miniprepped. The samples were stored in elution buffer (10mM Tris-HCl, pH 8.0) in the 4°C. A shortage of QIAprep Mini Columns was discovered in the lab, rendering these minipreps more time-consuming than usual. The first successful (out of seven previous attempted) gel was run on the a. niger gDNA.

  June 25

  • The day was devoted to running a PCR cycle in order to isolate the carrot extensin gene. In a 50µL reaction, the following volumes of reagents were used: 5µL 10X Standard Taq Reaction Buffer; 1µL 10mM dNTP mix; 0.5µL 20mM upstream primer; 0.5µL 20mM downstream primer; 7.31µL DNA template at 68.4ng/µL; 0.25µL Taq DNA Polymerase; and 35.44µL DEPC-treated water. Initial denaturation of 5 minutes at 95°C, followed by 30 cycles of 95°C (30 seconds), 73°C (60 seconds), and 68°C for 60 seconds, and a final extension at 68°C for 5 minutes. The PCR was unsuccessful, as our phosphorimage shows.

  June 26

  • A second PCR cycle in order to isolate the carrot extensin gene was attempted; a gel showed that the PCR was, again, unsuccessful. The following five treatments were placed in the 37°C as an O/N culture: LB control; LB, DH5α, ampicillin; LB, DH5α, chloramphenicol; LB, DH5α, chloramphenicol, K652001; LB DH5α, ampicillin, pSB1A3; and Terrific Broth, DH5α, ampicillin, and pSB1A3.The A. thaliana DNA was lyophilized in preparation for the plant gDNA miniprep.

  June 27

  • Ligation of the Kozak sequence to pSB1A3, and research into the melting temperatures of the primers for HSP18.2, AtPAP15, and PHT1;2.

  June 28

  • Isolated A. thaliana DNA-3.6 ng/ul and 3/7 ng/ul (double the value of yesterday); precut before freezing + centrifuged to prevent viscous lysates from shearing DNA apart
  • Transformed psb1A3 + kozak ligations into competent cells, plated and left to grow overnight

  June 29

  • PCR of HSP18.2 performed with A.Thaliana DNA to isolate gene with restriction sites.
  • Gel run with PCR products indicates that the PCR has failed, no visible bands.
  • Selected 4 white colonies from PSB1A3 + kozak transformation, growing up with ampicillin.

  June 30

  • Miniprepped 4 kozak colonies (names koz 1-4) from overnight culture. Koz 2 was found to be red after spinning down, and was discarded. Other 3 samples were miniprepped and nanodropped. Koz 1: 110.9 ng/ul; Koz3: 118.8 ng/ul; Koz4: 47.6 ng/ul;
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