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Team:Queens Canada/Notebook/Week8
From 2012.igem.org
Notebook - Week 8
| Date | Protocol | People | DNA (if relevant) | Quantities and Parameters (if relevant) | Notes on Protocol/ Gel Lanes |
|---|---|---|---|---|---|
| Jun-17 | Liquid Culture | Kevin, Beini | BL21 Cells | ||
| Jun-18 | PCR | Beini, Victor, Andrew | Lead binding Protein, GFP w/ linkers, CFP | Mastermix ddH2O 10 ul x 9 = 90 uL Hotstart 12.5 uL x 9 = 112.5 uL LP 0.75 uL RP 0.75 uL Template 1 uL Total 25uL Program:1. Denaturation (Initial): 95 for 3 mins 2. Denaturation: 95C for 20 seconds 3. Annealing: 60C for 15 seconds 4. Extension: 72 degrees, 20 seconds 5. Repeat step 2-4 for 20 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | |
| Kapa Readymix PCR | David, Kevin, Dan | FliC C1 PCR Mastermix for 1/2 Colony PCR x 4 | Mastermix Kapa Readymix 50 uL F primer: 3 uL R primer: 3 uL Colonies: 4 uL Water: 40 uL Total : 100 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, 20 seconds 5. Repeat step 2-4 for 30 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | |
| Mastermix for 10 uL liquid colony x4 | Mastermix Kapa Readymix 50 uL F primer: 3 uL R primer: 3 uL Liquid Culture: 40 uL Water: 4 uL Total : 100 uL | ||||
| Mastermix for 5 uL liquid culture x 2 | Mastermix Kapa: 25 uL F primer: 1.5 uL R primer: 1.5 uL Liquid Culture: 10 uL Water: 12 uL Total: 50 uL | ||||
| Control Mastermix x2 | Mastermix Kapa: 25 uL F primer: 1.5 uL R primer: 1.5 uL Water: 22 uL Total: 50 uL | ||||
| Double Gel Recipe | 14 x 2 Lanes, a little bit bubbly | Gel: 1) Ladder, 2) PbrC, 3) Pbr1, 4) Pbr2, 5) GC, 6) G1, 7) G2 8) Ladder, 9) CFP C, 10) CFP 1, 11) CFP 2, 12) Control , 13) PC, 14) Blank, 15) Ladder, 16) P1, 17) P2, 18)P3, 19) P4, 20) Ladder, 21) L1, 22) L2, 23) L3, 24) L4, 25) L5, 26) L6, 27)Blank | |||
| Jun-19 | Received biobrick parks | Got Biobrick ordered parts (31 of them) had 27 LB plates so plated 24 part on chloramphenicol using sterile loop and agar stab plated 3 parts on ampicillinn plates using sterile loop and agar stab had 4 parts left over (3 amp, 1 specR) placed the 27 streaked plates in the 37 degrees celcius incubator, placed the used agar stabs in the 4 degrees celcius fridge with the 4 unused agar stabs sitting at on the lab bench | |||
| LB Plates | Victor, Andrew | 40 Plates were made, refer to Agar Plate Protocol | |||
| Jun-20 | PCR | Victor, David, Faisal, Beini | 8 Tubes Label L1, L2 - Liquid colony normal left primer C1, C2 - One colony normal left primer LP1, LP2 - Liquid Colony w/ Promoter CP1, CP2 - One colony w/ Promoter Ctrl, Ctrl P - Control, Control w/ Promoter | PCR Mastermix dd H20 10 uL Hotstart 12.5 uL LR 0.75 uL RP 0.75 uL Template 1 uL Total 25 uL | Gel: 1) 1kB+, 2) CtrlP, 3) L1, 4) L2, 5) Lp1, 6) Lp2, 7) Blank 8) Blank, 9) Blank C, 10) Ctrl , 11) C1 , 12) C2, 13) Cp1, 14) Cp2, 15) Blank, 16) Blank, |
| Jun-21 | Miniprep | Faisal, Beini, Phillip, Victor | |||
| Liquid Culture | |||||
| PCR/Gel | Faisal, Beini, Phillip, Victor | FliC Parts ( 5 uL of template), PCR 14 trials total. Templates : mCitr (323 062) mCer (323 023), N-split Citr (323 072), C-split Citr (323 055), N-split ceru (323 011), C-split ceru (323 012) | PCR Mastermix dd H20 10 uL Hotstart 12.5 uL LR 0.75 uL RP 0.75 uL Template 5 uL Total 25 uL Program:1. Denaturation (Initial): 95 for 2:30 mins 2. Denaturation: 98C for 20 seconds 3. Annealing: 60C for 15 seconds 4. Extension: 72 degrees, 15 seconds 5. Repeat step 2-4 for 15 cycles 6. Final extension 72 degrees, 3 minute 7. Cool down 4 degrees, one hour | Double Recipe Gel: 1) Ladder, 2)FliC Control, 3) Liquid Culture Trial 1, 4) Liquid Culture Trial 2, 5) Colony Trial 1, 6) Colony Trial 2, 7) Ladder, 8) C- split Control, 9) C-split Trial 1 (citrine), 10) C-split Trial 2 (citrine), 11) C-split Trial 1 (Cerulean), 12) C-split Trial 2 (cerulean), 13) Blank, 14) Blank, 15) Ladder, 16) Full Control, 17) Full Citrine Trial 1, 18) Full Citrine Trial 2, 19) Full Cerulean Trial 1, 20) Full Cerulean Trial 2, 21) Ladder, 22) N-split Control, 23) N-split Trial 1 (Citrine), 24) N-split trial 2 (Citrine), 25) N-split Trial 1 (cerulean), 26) N-split Trial 2 (cerulean) 27) Blank, 28) Blank | |
| Jun-22 | PCR/ Gel | David, Faisal, Beini | mCerulean K323023, mCitrine K323062, N-split mCer K323011, C-split mCer K323012, N-split mCit K323072, CFP Series Control, N-split series control. Primers: CFP Series, Csplit FP_LP, Nsplit FP_RP, CFP_LP, N-split FP_RP | Mastermix Kapa: 12.5 uL F primer: 0.75 uL R primer: 0.75 uL Water: 10 uL Template DNA: 1 uL Total: 25 uL Program:1. Denaturation (Initial): 95 for 5 mins 2. Denaturation: 98C for 30 seconds 3. Annealing: 55C for 15 seconds 4. Extension: 72 degrees, 35 seconds 5. Repeat step 2-4 for 30 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | Gel 1) Ladder, 2) C.C, 3) mCer1, 4) mCer2 5) mCit 1, 6) mCit 2, 7) NmCer1 8)NmCer2 9) Ladder, 10) NC, 11) CmCer1 12)CmCer2 13) NmCit 1, 14) NmCit 2. |
| Plate Streaking | Kevin | BL21 Competent Cells. |
Monday June 18
In the morning, we looked at different protocols for colony PCR and from those, designed our own protocols, hoping at least one of them would work to amplify our fliC gene. Andrew, Victor, and Beini went into the lab to do PCR of fliC1, GFP, CFP, and PbrR. In the afternoon, Kevin brought in a delicately moist carrot cake topped with a smooth, rich, cream cheese-based icing. After everyone on the team had their share, we brought it over to the Chin-Sang lab for them to dig in. Aaron, David, Dan, and Kevin went into the lab to do colony PCR (which proved to be quite mind-numbing) and make a super gel of our latest PCR products. The rest of us spent the afternoon working on sponsorship and reading about chimeric restriction endonucleases.
Tuesday June 19
This morning we received our second shipment of BioBrick parts in the form of 31 agar stabs. David, Phillip, and Beini worked on inoculating all 27 of the plates we had left with our parts, while Victor and Andrew worked on making more LB plates. Dan and Kevin did more research on chimeric restriction enzymes. After lunch, we all sat down to put our brains together to evaluate the feasibility of making our own chimeric restriction enzymes. Seeing as our deliberation of BioBrick fusion standards has dragged on long enough, Kevin devised a way to decide which one we are ultimately going to use: everyone picks a standard, formulates a convincing argument for why we should use that standard, and then we show and tell! We only had time for David to present his case on the Silver fusion standard (RFC 23), which he did with no shortage of finesse, but we will continue this exciting debate tomorrow!
Wednesday June 20
This morning, we finished off the fusion standard mini-presentations. The final verdict? Tom Knight’s BB-2 standard (RFC 12) would be the most ideal for our purposes. It would be neat for us to test it out to see if it works, since it hasn’t been widely adopted by the synthetic biology community (yet). In the afternoon, Kevin and Maggie played around with Gromacs, a protein modelling software that Dr. Campbell recommended. In the lab, David, Victor, Faisal, and Beini had an assembly line going to prepare 31 liquid cultures. They also tried colony PCR using a different method lent to them by Jun (a Ph.D. student in the Chin-Sang lab).
Thursday June 21
In the morning, we searched the web for genomic DNA isolation protocols to use for our colony PCR. So far, we have not yet been successful in amplifying our fliC constant domain 1 gene, which is more than a little irksome. In the lab, we tried colony PCR with 5 ul of template DNA rather than our usual 1 ul. Faisal and Phillip spent ~2 hours laboriously labelling 31 Eppendorf tubes and spin columns in preparation for mini-preps galore. After lunch, we compiled a summary document of all the BioBricks we have ordered to date, to keep track of our progress for each part. We also discovered that the newspaper article about us got posted to the Kingston This Week website, featuring an excellent photo of Phillip, Andrew, and Kevin looking very happily down at a plate that spelled out QGEM in RFP bacteria. However, there was a typo with Andrew’s last name, spelled as “Paham” (now he can’t even show the article to his Phamily, har har har).
Friday June 22
Today, we ran a PCR and gel of five parts with similar annealing temperatures: mCerulean, mCitrine, and variations. We lowered the melting temperature and raised the number and length of cycles, hoping to increase the sensitivity of our products. These parameters seemed to work well, although we did get some unwanted products on the gel. While Faisal was making the Master Mix, he noticed a large particle floating around in our bottle of ddH2O, reminiscent of newt poo... time to get a fresh bottle. We made a few errors while we carried out the PCR, but always caught them in time... Friday labwork is the most fun! However, we are running out of Hot Start polymerase, so we can’t afford to make too many silly errors. Kevin had a meeting with Melissa Mahady Wilton from the Kingston School of Dance about our SynthetiQ project. She might be able to help us get an artist-in-residence, which would greatly facilitate the actual creation of our dances.
