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Team:Queens Canada/Notebook/Week13
From 2012.igem.org
Control
Notebook - Week 13
| Date | Protocol | People | DNA (if relevant) | Quantities and Parameters (if relevant) | Notes on Protocol/ Gel Lanes |
|---|---|---|---|---|---|
| Jul-23 | Gel | Andrew | Gel: 1) 1 KB+ Ladder, 2)pfliC1 T1, 3)pfliC1 T2, 4)pfliC1 T3, 5)pfliC1 + GFP1, 6)pfliC1 + GFP2, 7) pfliC1 +GoIB 1, 8) pfliC1 + GoIB2 | ||
| Rehydration of primers | Phillip | Primers not specified | |||
| Gel | Phillip | Gel: 1) Ladder, 2) pFliC1 + fliC2 + GFP1 C, 3) pFliC1 + fliC2 + GFP1 1, 4) pFliC1 + fliC2 + GFP1 2, 5) pFliC1 + fliC2 + GFP2 C, 6) pFliC1 + fliC2 + GFP2 T1, 7) pFliC1 + fliC2 + GFP2 T2, 8) Ladder, 9) pFliC1 + fliC2 + GFP3 C, 10) pFliC1 + fliC2 + GFP3 T1, 11) pFliC1 + fliC2 + GFP3 T2 | |||
| Gel Extraction | pfliC 1 (3 trials), pfliC1 + GFP 1 and pfliC 1+ GFP 2 | ||||
| Dry mix of soft agar prepared | [TET] | ||||
| Jul-24 | PCR Purification | Followed standard NEB protocol for PCR purification for: pFliC1 + fliC2 + GFP2 (C, T1 and T2) | Thermopol Protocol: Thermopol: 5 ul dNTPs: 1 ul Template: 10 ul LP fliC1: 1.5 ul RP fliC2: 1.5 ul Vent Pol: 0.7 ul MgSO4: 1 ul H2O: 28.3 ul Total: 33.3 ul Using LP of fliC C1 and RP of respective part. Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 95 C for 20 seconds 3. Annealing: 72 C for 15 seconds 4. Extension: 72 degrees, 120 seconds 5. Repeat step 2-4 for 20 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | ||
| Gel Extraction | David and Faisal | pFliC + GFP GE and pfliC GE | Gel: 1) Ladder, 2) pfliC + GFP GE T1, 3) pfliC + GFP GE T2, 4) pfliC GE Trial 1, 5) pfliC GE Trial 2, 6) pfliC GE Trial 3, 7) PfliC GE Trial 4, 8) pfliC GE Trial 1 | ||
| Motility Assays | Beini | TOP 10 [STREP] | |||
| Soft Agar Plates | Beini | Made five soft agar plates with [Tet] | |||
| Gel | Phillip | Gel: 1) Ladder, 2) pfliC1 + fliC2 +GFP 2 C, 3) pfliC 1 + fliC2 + GFP 2 T1, 4) pfliC1 + fliC2+ GFP 2 T2, 5) Ladder | |||
| Gel | Faisal, David | Gel: 1) Ladder, 2) pfliC + GFP GE Trial 1, 3) pfliC + GFP GE Trial 2, 4) pfliC GE Trial 1, 5) pfliC GE Trial 2, 6) pfliC GE Trial 3, 7) Ladder, 8) pfliC GE Trial 4, 9) fliC C1 GFP GE Trial 1, 10) fliC C1 GFP GE Trial 2. | |||
| PCR | Phillip | pfliC + GFP GE + FliC2 ( six trials) | Thermopol Protocol: Thermopol: 5 ul dNTPs: 1 ul fliC2 : 5 ul pfliC + GFP : 5 ul Vent Pol: 0.7 ul MgSO4: 2 ul H2O: 31.5 ul Using LP of fliC C1 and RP of respective part. Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 95 C for 20 seconds 3. Annealing: 65 C for 15 seconds 4. Extension: 72 degrees, 90 seconds 5. Repeat step 2-4 for 30 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | ||
| Digestion | Phillip | pFliC + GFP GE T1 + T2 digested w/ Fermentas fast digest SpeI | Used the standard Fermentas Fast Digest Protocol | ||
| Jul-25 | Gel | Andrew | Gel: 1) 1 KB+, 2) pfliC1 + GFP GE T1 + FliC2 dig T1, 3) pfliC1 + GFP GE T1 + FliC2 dig T2, 3) pfliC1 + GFP GE T1 + FliC2 dig T3, 4) pfliC1 + GFP GE T2 + FliC2 dig T1, 5) pfliC1 + GFP GE T2 + FliC2 dig T2, 6) pfliC1 + GFP GE T2 + FliC2 dig T3 | ||
| Motility Results | Beini | Small circles, motility assay of XLI Blue on [TET] plates. | |||
| Jul-26 | Uber Gel | Beini | Gel: 1) 1 KB+, 2) GFP + pSB1C3 T3 LIG, 3) GFP + pSB1C3 MPP, 4) E0040 GFP MPP T1, 5) fliC2 T1 XS DIG - nothing, not enough in tube, 6) fliC2 T2 XS Dig, 7) fliC2 EP NEB DIG, 8) 1 KB+, 9) fliC2 EP FD DIG, 10) fliC2 + pSB1C3, 11) GFP T2 + fliC2 + pSB1C3, 12) med RBS +fliC1 T2, 13) med RBS + fliC1 T3, 14) med RBS + fliC T4, 15) 1 KB+, 16) med RBS + fliC1 T2 MPP, 17) med RBS + fliC1 T3 MPP, 18) med RBS + fliC1 T4 MPP, 18) med RBS + fliC1 T4 MPP, 19) Strong RBS + fliC1 T1, 20) Strong RBS + fliC1 T2, 21) Strong RBS + fliC1 T3, 22) 1 KB+ Ladder, 23) Strong RBS + fliC1 T1 MPP, 24) Strong RBS + fliC1 T2 MPP< 25) Strong RBS + fliC1 T3 MPP, 26) (Med RBS+ fliC1) + fliC2 +term T1, 27) (med RBS +fliC1) + fliC2 + term T2, 28) (Strong RBS +fliC1) + fliC2 + term T3 | ||
| PCR | Philiip | Took PCR that I did on 07/24 and added 5 ul pfliC1 and 1.5 ul fliC2 Rp? (HARD TO READ) | Hotstart PCR Protocol: Hotstart 12.5 ul FP (fliC1) 0.75 ul RP (GFP) 0.75 ul FliC1 3 ul GFP 2 ul ddH20 6 ul Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 95 C for 20 seconds 3. Annealing: 65 C for 15 seconds 4. Extension: 72 degrees, 90 seconds 5. Repeat step 2-4 for 30 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | Gel Electrophoresis 1) Ladder, 2) pfliC + GFP GE T1 + fliC 2 dig T1, 3) pfliC + GFP GE T1 + fliC 2 dig T2, 4) pfliC + GFP GE T1 + fliC 2 dig T3, 5) pfliC + GFP GE T2 + fliC 2 dig T1, 6) pfliC + GFP GE T2 + fliC 2 dig T2, 7) pfliC + GFP GE T2 + fliC 2 dig T3 | |
| PCR Purification | Phillip | pfliC GE GFP PCR Purification (5 trials) | |||
| Gel Extraction | Beini, David | From 07/26 Uber Gel Lane 6 FliC C2 T2 X+S Digest | |||
| Organized | Beini | Organized digestions/ ligations box by date 07/03 - 07/24 | |||
| PCR | FliC2 + GFP PP ( 1 control and three trials) | Hotstart PCR Protocol: Hotstart 12.5 ul FP (fliC1) 0.75 ul RP (GFP) 0.75 ul FliC1 3 ul GFP 2ul ddH20 6 ul Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 95 C for 20 seconds 3. Annealing: 63 C for 15 seconds 4. Extension: 72 degrees, 80 seconds 5. Repeat step 2-4 for 25 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour |
Given that the vast majority of our work now is in the lab, we will only be updating the "Labwork" section of the Notebook.
