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Team:Queens Canada/Notebook/Week10
From 2012.igem.org
Notebook - Week 10
| Date | Protocol | People | DNA (if relevant) | Quantities and Parameters (if relevant) | Notes on Protocol/ Gel Lanes |
|---|---|---|---|---|---|
| Jul-03 | Digestion and Ligation | Dan, Victor, Beini | pSB1C3 and GFP + fliC2 pSB1C3 | Digestion Mix: (GFP/fliC2) Eco RI - HF / XbaI SpeI / PstI 10X NEB Buffer 2 100X BSA H20 | |
| Streak Plates of competent cells | BL21 DE3 plyss [C] 2 trials XLI Blue [tet] 2 trials DH5 - alpha [nal] 2 trials | ||||
| Heat shock transformation | Dan, | FliC C2 in Kanamycin | |||
| Ligation | David | fmT PCR product into plasmid 1A3 | Ligation Mix: 2 ul PCR Product 1 ul EcoRI 1 ul PstI 5 ul NEBuffer 2 0.5 BSA 40.5 Water Plasmid: 4 ul plasmid + 4 ul mastermix | ||
| Heat shock transformation | Andrew | 5 parts: GFP + fliC C2 + psB1C3 (two trials) GFP + pSB1C3 fliC2 + pSB1C3 fmt + pSB1A3 | |||
| Jul-04 | Digestion and Ligation | 1)GFP, 2) GFP, 3) GFP, 1) fmT, 2) fmT, 3) fmT | Digestion Protocol (NEB): NEB2: 2.5 ul BSA: 0.5 ul EcoRI: 0.5 ul PstI : 0.5 ul Used parts registry protocol for digestion: 1)GFP: DNA 10 ul, H20 6 ul, 2) GFP: DNA 5 ul, H20: 11 ul 3)GFP: DNA 2 ul, H20 14 ul, 1) fmT: DNA 10 ul, H20: 6 ul, 2)fmt: DNA 5 ul, H20 11 ul, 3) fmT: DNA 2 ul, H20 14 ul | ||
| Liquid Cultures | XLI Blue, BL21 DE5 Plys, DH5α, 2x fliC kanamycin, | ||||
| Ligations | 6 Ligations: 1) GFP + pSB1C3 ligation T1 2) GFP + pSB1C3 ligation T2 3) GFP + pSB1C3 ligation T3 4) fmt + pSB1A3 ligation T1 5) fmt + pSB1A3 ligation T2 6) fmt + pSB1A3 ligation T3 | Protocol was not recorded, NEB protocol was used. | |||
| Jul-05 | Miniprep | Victor, Kevin, Beini | fliC C2 x3 | ||
| Genomic DNA Isolation | XLI Blue, BL21 DE5 Plys, DH5α. | 70% Ethanol required for the protocol was diluted using 85% Ethanol. | |||
| Primer Resuspension and dilutions | David | Metal-Binding Part primers: Primer resuspension and dilutions for GoIB, SmtA and MBP | note: MBP LP may not have exact amount of TE added during resuspension due to pipetting error. Note: SmTA LP dilution may not have enough primer added --> If PCR failts try making another dilution | ||
| Jul-06 | PCR | David, Andrew | PbrR, SmtA, GolB Four trials of each + one control each | Mastermix Thermopol Rxn Buffer: 5 ul DNTPs (10 mM): 1 ul LP: 1.5 ul RP: 1.5 ul DNA Template: 5 ul Vent Polymerase: 0.5 ul MgSO4: 1 ul H2O: 34.5 ul Total: 50 ul Program:1. Denaturation (Initial): 95 for 5 mins 2. Denaturation: 95C for 20 seconds 3. Annealing: 60C for 15 seconds 4. Extension: 72 degrees, 30 seconds 5. Repeat step 2-4 for 30 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | Big Gel: 1) Ladder, 2)mCit FD digest EP, 3) PbrR Control 4) PbrR Trial 1, 5) PbrR Trial 2, 6) PbrR Trial 3, 7) PbrR Trial 4, 8) Ladder, 9) SmtA Control, 10) SmtA Trial 1, 11) SmtA Trial 2, 12) SmtA Trial 3, 13) SmtA Trial 4, 14) SmtA Trial 5, 15) Ladder, 16) GoIB Control, 17) GoIB Trial 1, 18) GoIB Trial 2, 19) GoIB Trial 3, 20) GoIB Trial 4, 21) GoIB Trial 5, 22) Ladder, 23) PbrR Trial 5. |
| NEB Protocol Digestion | Victor | XLI Blue, BL21 DE5 Plys, DH5α, fliC C2, Luciferase, mCitrine, | Small Gel: 1) Ladder, 2) DH5α NEB EP Digest,3) DH5α Control, 4) XLI Control, 5) LI Digest Blue NEB EP, 6) BL21 Control, 7) BL21 Fast Digest EP, 8) fliC C3 Digest NEB EP, 9) Ladder,10) fliC C2 Control, 11) fliC C2 digest FD EP, 12) Luciferase Control, 13) Luciferase NEB T1 EP, 14) Luciferase NEB T2 EP, 15) mCitrine Control, 16) mCit FD Digest EP. | ||
| Heat Shock Transformation | Andrew | Parts list BBa_: J04500 - Promoter, J13002 - Promoter, 1712074 - Promoter, B0010 - Terminator, B0015 - Terminator, B0017 - Terminator, B0030 - Strong RBS, B0031 - Weak RBS, B0032 - Medium RBS | |||
| Jul-06 | PCR (Thermopol Protocol) | Kevin | Four trials of each strain + one control | Thermopol Protocol: Thermopol: 5 ul dNTPs: 1 ul LP: 1.5 ul RP: 1.5 ul DNA Template: 5 ul Vent Pol: 0.5 ul MgSO4: 1 ul H2O: 34.5 ul Program:1. Denaturation (Initial): 95 for 5 mins 2. Denaturation: 98C for 20 seconds 3. Annealing: 55C for 15 seconds 4. Extension: 72 degrees, 30 seconds 5. Repeat step 2-4 for 30 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | |
| Jul-07 | Miniprep | Kevin, Maggie | fmT+ pSB1C3, GFP + pSB1C3 (2 trials each) | ||
| Digestions | Using Trial #1 from above minipreps | NEB Digestion Protocol: 2 ul Templates 1 ul EcoRI HF 1 ul SpeI (NEB) 5 ul NEB Buffer 2 0.5 BSA 40.5 H20 Fast Digest Protocol: 2 ul Template 2 ul 10x FD Buffer Green 1 ul SpeI (FD) 1 ul EcoRI (NEB) 14 ul H20 | |||
| Gel Electrophoresis | 28 Lanes total | Large Gel: 1) Ladder, 2) Control (fliC), 3) XLI Blue T1 4) XLI Blue T2, 5) XLI Blue T3, 6) XLI Blue T4 7) Ladder, 8) DH5α T1, 9) DH5α T2, 10) DH5α T3, 11) DH5α T4, 12) Blank, 13) Blank, 14) Blank, 15) Ladder, 16) BL 21 T1, 17) BL 21 T2, 18) BL 21 T3, 19) BL 21 T4, 20) fmT mpp T1, 21) Ladder, 22) fmT NEB, 23) fmT FD (Green) 24) GFP Mpp, 28) GFP NEB, 29) GFP FD (Green) | |||
| Jul-08 | PCR | Kevin, Maggie | XL1 Blue | PCR using XLI Blue: Buffer 5 ul DNTPs 1 ul LP 1.5 ul Rp 1.5 ul Template 5 ul Vent 0.5 ul MgSO4 1 ul H20 34.5 ul Program:1. Denaturation (Initial): 95 for 5 mins 2. Denaturation: 98C for 20 seconds 3. Annealing: 55C for 15 seconds 4. Extension: 72 degrees, 30 seconds 5. Repeat step 2-4 for 10 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | |
| PCR (Retry) / Gel | Kevin | PCR Retry using XLI Blue: Buffer 5 ul DNTPs 1 ul LP 1.5 ul Rp 1.5 ul Template 10 ul Vent 0.7 ul MgSO4 2 ul H20 28.3 ul Program:1. Denaturation (Initial): 95 for 5 mins 2. Denaturation: 98C for 20 seconds 3. Annealing: 54C for 15 seconds 4. Extension: 72 degrees, 30 seconds 5. Repeat step 2-4 for 36 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | Gel of FliC w/ from XLI Blue: 1) Ladder, 2) XL1 T1, 3) XL1 T2, 4) XL1 Control, 5) Ladder 6)XLIB 1, 7) XLIB 2, 8) XLIB 3 | ||
| Miniprep | Kevin | B0015 - Double Terminator Trial 1, J04500 - T7 Promotoer (Two Trials), B0011 - Weak RBS (Two Trials), BB030 - Strong RBS (Two Trials), 2 Trials of the rest of the parts from Jul-5 except double terminator 2 (B0017, only one trial) | For next time, only use 1 mL of culture so that there's less stuff after adding solution 3. Easier to pipette supernatent |
Tuesday July 3rd
To start the day, we examined the gels we ran on Friday to try to decide our next plan of attack for isolating fliC C1. Should we try isolating genomic DNA of BL21 again, perhaps using a different protocol that doesn’t involve spooling the DNA out with a Pasteur pipette? Or should we abandon BL21 altogether and try a different strain, such as HB101? It might be a good idea to set a deadline for isolating fliC C1, and just get the gene synthesized if we are unable to do so by that date. Throughout the day, we continued research in our respective sub-groups, and significant progress was made in the Cohesins and Dockerins group. In the lab, we tried some digestions and ligations of parts with fliC C2, and plated competent cells of BL21 DE3 pLysS, XL1 Blue, and DH5-alpha. After lunch, Kevin brought in mini Canada Day-themed cupcakes for everyone.
Wednesday July 4th
All the ligations from yesterday failed, unfortunately. In the lab this morning, Victor and David tried ligations of fmT and GFP with fliC C2. In the afternoon, Victor and Phillip grew liquid cultures for fliC C2 and the three different strains of E. coli that were plated yesterday. As for research topics, Kevin, Dan, and Andrew hunted for gene sequences for specific cohesins and dockerins. Phillip worked on haloacetate degradation and Beini worked on polymerization of flagellin subunits.
Thursday July 5th
Morning newsflash from Andrew: the incubator was set to 54°C. Our latest digestion-ligations didn’t work... now we don’t know whether it was because of the cranked up incubator or some other reason. For the future, we should either use the 37°C incubator room, or just be more careful about brushing the knob of the incubator. A whole bunch of emails were sent today: contacting sponsors, ordering enzymes, requesting genes/plasmids from investigators, etc. Phillip updated the scroll to top button on our wiki. In the afternoon, we received a package from our sponsor Novus Biologicals, containing cool gear like antibody-shaped floaters in fun colours, highlighter-pens, lanyards, and fridge magnets. Our liquid cultures from yesterday worked, so we miniprepped those (fliC C2 + three strains of E. coli). We also attempted a second genomic DNA isolation, this time using a slightly different protocol that did not involve spooling the DNA out and transferring it to another tube. We did this for three strains of E. coli (XL1 Blue, DH5-alpha, and BL21 DE3 pLysS). Those involved in SynthetiQ met tonight and did some bodystorming for our upcoming PCR video, and well as some brainstorming about logistics. We decided that it would be ideal to have 8 dancers involved to effectively illustrate DNA concepts.
Friday July 6th
The lab was buzzing with activity today as we are in full swing with our digestions/ligations and PCRs. Victor, David, Andrew, and Phillip worked in the lab for most of the day, while Kevin and Beini researched the construction of fliD chimeras and motility assays. After lunch, Phillip brightened everyone’s day with chocolate chip cookies that he baked himself. They disappeared pretty quickly. Beini tried designing primers to amplify the fliD constant sequences, but the primers aren’t very ideal, so if we are planning to make fliD chimeras at all, we may want to consider getting the two constant sequences synthesized.
