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Team:Queens Canada/Notebook/Week14
From 2012.igem.org
Control
Notebook - Week 14
| Date | Protocol | People | DNA (if relevant) | Quantities and Parameters (if relevant) | Notes on Protocol/ Gel Lanes |
|---|---|---|---|---|---|
| Jul-27 | Gel | Gel: 1) Ladder, 2) PP GFP T1, 3) PP GFP T2, 4) PP GFP T4, 5) GFP Dig T1 XS, 6) GFP Dig T2 XS, 7) GE fliC2 T2 XS, 8- 14 are blank, 15) 1 KB+ Ladder, 16) pF GE 1 GFP1 C, 17)pF GE 1 GFP1 T1, 17) pF GE 1 GFP2 T1, 18) pF GE 2 GFP1 T1, 19)pF GE 2 GFP2 T1, 20) pF GE2 GFP 2 T1, 21) F2 GFP PP C, 23) f2 GFP PP T1, 22) 1 KB+, 24) F2 GFP PP T2, 25) F2 GFP PP T4, 26) Blank, 27) GFP XS DP T2, 28) GE FLiC2 T2 XS. | |||
| PCR | Phillip | pFliC GFP PP ( 1 control and 4 trials) | Hotstart PCR Protocol: Hotstart 12.5 ul FP (pfliC) 0.75 ul RP (GFP) 0.75 ul FliC1 3 ul GFP 2ul ddH20 6 ul Program Not Recorded | ||
| PCR | Phillip | pFLiC GE (1 control, 4 trials) | Hotstart PCR Protocol: Hotstart 12.5 ul FP (pfliC) 0.75 ul RP (GFP) 0.75 ul FliC1 3 ul GFP 2ul ddH20 6 ul Program Not Recorded | ||
| Liquid Cultures | Beini | XLI Blue [Tet] | |||
| Motility Plate | Beini | XLI Blue [Tet] | |||
| Heat Shock Transformation | Beini | fliC C2, MPP T | |||
| Gel Electrophoresis | Philip | Gel: 1) Ladder, 2)pfliC GEC , 3) pfliC GE T1, 4) pfliC GE T2, 5)pfliC GE T3, 6), pfliC GE T4, 7) Blank, 8) Blank, Lane 2: 1) Ladder, 2) pfliC Gel Extraction PCR Purification T1, 3)pfliC Gel Extraction PCR Purification T2, 4) pfliC Gel Extraction PCR Purification T3, 5) pfliC Gel Extraction PCR Purification T4, 6)pfliC Gel Extraction PCR Purification T | |||
| Gel Extraction | David | F2 GFP PP T4. F2 GFP PP T1, pF GE2 GFP 1 T1, pf GE2 GFP2 T1 | |||
| PCR | Faisal, David | XyIE | Hotstart PCR Protocol: Hotstart 12.5 ul FP (fliC1) 0.75 ul RP (GFP) 0.75 ul Template 1,2,3 ul ddH20 8 ul Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 98 C for 20 seconds 3. Annealing: 60 C 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 | ||
| Soft Agar Antibiotic Plates | |||||
| Digestion | FliC2 + GFP and pFliC + GFP 2 (Two Trials of each) | ||||
| PCR Overlap Extension | pFliC FliC1 - GFP- S, X - GFP - FLiC 2 | Hotstart PCR Protocol: Hotstart 12.5 ul FP (fliC1) 0.75 ul RP (GFP) 0.75 ul Template 2 ul ddH20 10 ul Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 98 C for 20 seconds 3. Annealing: 61 C for 15 seconds 4. Extension: 72 degrees, 2 min 5. Repeat step 2-4 for 25 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | |||
| Soft Agar Plates | Beini | ||||
| Jul-28 | Motility Plate Check | Kevin | GFP | ||
| Gel | Gel: 1) 1 KB+ , 2) FF GFP 1, 3) FF GFP 2 dim, 4) FF GFP 3, 5) FF GFP 4, 6) XyLE 1, 7) 1 KB+, 8) XylE 2, 9) XyIE 3, 10) Blank, 11) XyIE C, 12) Blank, 13) 1 KB+, 14) pfliC GE PCR C, 15) pfliC GE PCR 1, 16) pfliC GE PCR 2, 17) pfliC GE PCR 3, 18) pfliC GE PCR 4, 19) flipp 1, 20) flipp 2, 21) 1 KB+, 22) Blank, 23) Blank, 24) Lane 19 Gel extract of FLiC1 + GFP, 25) Above, dig S, 16) Above DP, 27) Lame 2S (FliC2 + GFP Extract), 28) DIg X, 29) DP | ||||
| Liquid Cultures | FliC2 (Three trials) | ||||
| Mobility Plate | XLI Blue | ||||
| Gel Extraction | FF GFP | ||||
| Gel Electrophoresis | Gel: 1) 1 KB+, 2) FF GFP 1, 3) FF GFP 2, 4) FF GFP 3, 5) FF GFP 4), 6) FF GFP Extract 2, 7) FF GFP Extract 4, 8) 1 KB+ | ||||
| Jul-29 | PCR Overlap Extension | David, Kevin | Adding promotor to RBS fliC | Hotstart PCR Protocol: Hotstart 12.5 ul FP (fliC1) 0.75 ul RP (GFP) 0.75 ul Template 3 ul ddH20 8 ul Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 98 C for 20 seconds 3. Annealing: 60 C for 15 seconds 4. Extension: 72 degrees, 1 min 5. Repeat step 2-4 for 25 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | |
| Motility Assay from Glycerol Stock | One for each stock, 1) DHS, 2) XLI Blue, 3) Top 10, 4) BL21. | Gel: 1) Ladder, 2) pFliC GFP 1, 3)pFliC GFP 2, 4)pFliC GFP 3, 5) pFliC GFP 4, 6) fliC2 Dig XS 1, 7)fliC2 Dig XS 2, 8) fliC2 Dig XS 3 Second Gel: 1) 1 KB+ Ladder, 2) pfliC GFP C, 3-7) Prom FliC C - 4, 8) Ladder, 9-12) pp Prom FliC 1-4 | |||
| Miniprep | David | MPP FliC C2 (Three Trials) | |||
| Gel Extraction | 07/29/2012 Lane 8 | ||||
| Glycerol Stock | FliC2 Plasmid from 07/27 plates and 07/28 liquid cultures | ||||
| Jul-30 | PCR Extension | David | RBS onto pFliC | Hotstart PCR Protocol: Hotstart 12.5 ul FP (fliC1) 0.75 ul RP (GFP) 0.75 ul Template 2 ul ddH20 9 ul Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 98 C for 20 seconds 3. Annealing: 60 C for 15 seconds 4. Extension: 72 degrees, 20 sec 5. Repeat step 2-4 for 20 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | |
| Motility AssayNotes | DHS, BL21, Top 10 and XLI Blue Glycerol Stocks | ||||
| Digestion | Victor | pFliC GFP (GE), pFliC GFP (GE), fliC2 GFP (GE), FliC 2 GFP (GE) T4 PCR extended | Gel: 1) Ladder, 2-4) XyIE T1- T3, 5-7), fliC 2 (XS) Lane 6- 8 GE | ||
| PCR | Victor Andrew | PCR Overlap Extension of pFliC + GFP and FliC2 + GFP (Four trials) | Hotstart PCR Protocol: Hotstart 12.5 ul FP (fliC1) 0.75 ul RP (GFP) 0.75 ul Template 2 ul ddH20 9 ul Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 98 C for 20 seconds 3. Annealing: 61 C for 15 seconds 4. Extension: 72 degrees, 20 sec 5. Repeat step 2-4 for 20 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | ||
| Liquid Cultures | XLI Blue, BL21, Top 10 | ||||
| Gel | Faisal | Gel: 1) Ladder, 2-6) RBS pfliC C-T4, 9) Ladder, 8-11) PP RBS + pfliC T1-T4 | |||
| Gel | Andrew | Gel: 1)Ladder, 2-6) pfliC + GFP + fliC2 Control-T4. New Gel: 1) Ladder, 2-7) DIG fliC1 T1-T7 | |||
| PCR | XyLE | Hotstart PCR Protocol: Hotstart 12.5 ul FP (fliC1) 0.75 ul RP (GFP) 0.75 ul Template 2 ul ddH20 9 ul Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 98 C for 20 seconds 3. Annealing: 55 C for 15 seconds 4. Extension: 72 degrees, 1 min 5. Repeat step 2-4 for 20 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | |||
| PCR | fliC Promotor | Hotstart PCR Protocol: Hotstart 12.5 ul FP (fliC1) 0.75 ul RP (GFP) 0.75 ul Template 5 ul ddH20 6 ul Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 98 C for 20 seconds 3. Annealing: 58 C for 15 seconds 4. Extension: 72 degrees, 30 sec 5. Repeat step 2-4 for 25 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | |||
| Gel | Andrew, Faisal | Gel 1) 1 KB+ Ladder, 2-5) XyIE Control-3, 6) Promo fliC Control, 7) Promo FliC1, 8) Ladder, 9-11) promo fliC 2-4 | |||
| Gel Extraction | Andrew | pFLiC GFP, fliC | |||
| Jul-31 | Mobility Plates | XLI Blue, BL21, XLI Blue Top 10 | |||
| PCR | David, Victor | pFliC GFP 1-4, C | Hotstart PCR Protocol: Hotstart 12.5 ul FP (fliC1) 0.75 ul RP (GFP) 0.75 ul Template 4 ul ddH20 7 ul Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 98 C for 20 seconds 3. Annealing: 61 C for 15 seconds 4. Extension: 72 degrees, 2 min 5. Repeat step 2-4 for 25 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | ||
| Digestion | David | DIG fliC2 GFP [X] 1,2, Lane 2, Lane 3 | |||
| PCR Purification | Promotor + flic (trials 1-4) EZ 10 Spin Column handbook Protocol and pFLiC Suffix 1-4 C | Hotstart PCR Protocol: Hotstart 12.5 ul FP (fliC1) 0.75 ul RP (GFP) 0.75 ul Template 4 ul ddH20 7 ul Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 98 C for 20 seconds 3. Annealing: 65 C for 15 seconds 4. Extension: 72 degrees, 20 sec 5. Repeat step 2-4 for 25 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | |||
| PCR Purification | Suffix-promoter- RBS fliC | Gel: 1)Ladder , 2-6) Suffix promo fliC1 C-4, 7-11) Suffix promo fliC1 1- 4, 12) MPP XyIE, 13) MPP RBS 0034, 14)MPP T7 Promo T1, 15) MPP T7 Promo T2 | |||
| Gel | Faisal | Gel: 1) Ladder, 2) pFLiC Suffix C, 3-7) pFliC GFP 1-4, C | |||
| PCR Overlap Extension | Victor | pfliC added Suffix 1-4, C | Hotstart PCR Protocol: Hotstart 12.5 ul FP (fliC1) 0.75 ul RP (GFP) 0.75 ul Template 2 ul ddH20 9 ul Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 98 C for 20 seconds 3. Annealing: 65 C for 15 seconds 4. Extension: 72 degrees, 20 sec 5. Repeat step 2-4 for 25 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | ||
| Gel Extraction | Beini | pfliC GFP FliC2 T3, T4 | Gel 1) 1 KB+ Ladder,2) pflIC1 + GFP + flIC2, 3) pfliC1 +GFP + fliC2 4-8) Suffix added FliC1- 4, 9) 1 KB+ | ||
| PCR Purification | Victor | pfliC Suffix 1-4 | |||
| Digestion | Faisal | FliC T1--> S+P, XyIE ---> X+P | Used standard Fast Digested Protocol | ||
| August-1st | PCR | Victor, David | pFliC+GFP+fliC2 Mastermix. PCR pfliC + added suffix LP + fliC2 RP ( 2 trials, one control) re pflIC GFP 1-4, C. | Hotstart PCR Protocol: Hotstart 12.5 ul FP (fliC1) 0.75 ul RP (GFP) 0.75 ul Template 2 ul ddH20 9 ul Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 98 C for 20 seconds 3. Annealing: 61 C for 15 seconds 4. Extension: 72 degrees, 20 sec 5. Repeat step 2-4 for 25 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | |
| Ligation | Faisal | Using NEB Protocol | |||
| Gel | Faisal | Gel 1) Ladder,2-3) RBS pfliC T1-T2, 4) promo + fliC1, 5) promo flic 1 PCR purification, 6) suff promo pfliC 2, 7) suffix promo pfliC 1 PCR purification, 8) pfliC added suffix 1, 9) pflic added suffix 1 PCR purification, 10) Ladder, 11) DIG T7 prom S+P, 12) DIg XyIE T1 X+P, 13) Dig T7 prom T2 S+P, 14) diG T7 prom T3 S+P, 15) Dig XyIE T3 X+P | |||
| Soft Agar Plates | Victor | Made 10 | |||
| Gel | Gel: 1) 1 KB+ Ladder, 2-6) re pfliC GFP 1-4, 6-8) adsuf pfliC GFP C, 1-2. | ||||
| Gel Extraction | Andrew | re pflic GFP | |||
| Gel | Gel: 1) 1 KB+, 2)-4) repfliC GFP Lane 3- 5, 5) 1 KB+ Ladder, 6) add suffix pflic GFP Lane 8, 7) Double terminator T2 (0015), 8) Double Terminator 0017T2. | ||||
| Digestions and Ligations | pFLiC GFP FliC2 into PSB1T3 | NEB Digestion Protocol: 2 ul Plasmid DNA 0.5 ul XbaI 0.5 ul Pst1 (NEB) 0.5 ul DRN1 2.5 ul NEB Buffer 2 0.5 BSA 14 H20 | |||
| Digestion | Digestion of repfliC GFP 2 GE with X and P (four trials) | Used the standard Fermentas Fast Digest Protocol | |||
| Ligation | LIG pfliC GFP fliC2 + pSBIT3 [Tet] T1A | ||||
| PCR | pfliC--- GFP ---- FliC2 | Hotstart PCR Protocol: Hotstart 12.5 ul FP (fliC1) 0.75 ul RP (GFP) 0.75 ul Template 2 ul ddH20 9 ul Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 98 C for 20 seconds 3. Annealing: 62 C for 15 seconds 4. Extension: 72 degrees, 2 min 5. Repeat step 2-4 for 25 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | |||
| Heat Shock Transformation | J45200 Banana Construct pSB1AT3 J33204 XyLE w/ RBS pSB1A2 K08806 Biotimer pSB1A2 K395602 Apple Scent pSB1C3 | ||||
| Gel | Gel: 1) 1 KB+, 2) re pfliC GFP 4, 3-6) ad suff. GFP C-3, 7) 1 KB+ Ladder, 8-11) pf GFP F2 C-3, 12) GFP 2 | ||||
| Aug-02 | PCR | Victor, David | Two constructs, three trials each: pfliC added LP + re pfliC GFP 2 GE + fliC 2 RP and asuf pFF GFP 1-3 | Hotstart PCR Protocol: Hotstart 12.5 ul FP (fliC1) 0.75 ul RP (GFP) 0.75 ul Template 2 ul ddH20 9 ul Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 98 C for 20 seconds 3. Annealing: 64 C for 15 seconds 4. Extension: 72 degrees, 1 min 5. Repeat step 2-4 for 25 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | |
| LB Plates | 21 of ampicillin, chloramphenicol, tetracycline and kanamycin | ||||
| Gel | Faisal | Gel: 1) KB+ ,2-5)LtG ff GFP 1T3 Control -T3, 6-8) pfGFP F2 1-3 | |||
| PCR | David | XyIE | Hotstart PCR Protocol: Hotstart 12.5 ul FP (fliC1) 0.75 ul RP (GFP) 0.75 ul Template 2 ul ddH20 9 ul Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 98 C for 20 seconds 3. Annealing: 60 C for 15 seconds 4. Extension: 72 degrees, 30 sec 5. Repeat step 2-4 for 25 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | ||
| Gel extraction | Victor | asuf IDT ffGFP 1-4, and asuf pFF GFP 1-3 | |||
| Gel Extraction | Beini | pF GFP F2 T1-T3 | |||
| Gel Extraction | Beini | adsuf IDT ff GFP 2-4, asuf pFF GFP 1-2 | Gel: 1) KB+ Ladder, 2-4) XyIE C-2, 5-7) GE pf GFP F2 T1-T3, 8) KB+ Ladder, 9) asuf pff GFP C, 10) asuf IDT ff GFP C, 11-13) GE asuf IDT ff GFP 2-4, 14-15) GE asuf pff GFP 1-2 | ||
| Digestion | pSB1C#, digested using X and P from yesterday's mastermix | ||||
| Ligations + Transformations | Faisal | plasmid backbone pSb1C3. | |||
| Liquid Cultures | J33204, XyIE + RBS and J48200, Banana Odor | ||||
| Liquid Cultures | David | LIG pflic GFP fliC 2 + pSBIT3, Ca, T3A, T2B and T1B, apple scent K395602, and bio- timer K088006 | |||
| Aug-03 | Miniprep | Victor | J33204 T1-T2, J48200 Banana odor | ||
| PCR | Victor | FliC1 No extensions ( Templates pfliC GE, LP: fliC1 LP, Rp : fliC RP | Hotstart PCR Protocol: Hotstart 12.5 ul FP (fliC1) 0.75 ul RP (GFP) 0.75 ul Template 2 ul ddH20 9 ul Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 98 C for 20 seconds 3. Annealing: 62 C for 15 seconds 4. Extension: 72 degrees, 30 sec 5. Repeat step 2-4 for 25 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | ||
| PCR Purifications | David | mCer1, mCer2, mCit1, mCit2, Nmcit1, Nmcit 2 all PCR Products from 06/22 | |||
| Digestion of PCR purification products | David | PP mCer1 with X+S, PP mCer 2 with X, PP mCit 1 with X+S, PP mCit 2 with X, PP Nm cit 1 with X+ S and PP Nmcit 2 with X | |||
| Rehydration and Transformations | Faisal | BBa_ I13602, I719015, I13601, E0430 | |||
| PCR | pFliC 1 with added base pairs (2 trials | Hotstart PCR Protocol: Hotstart 12.5 ul FP (fliC1) 0.75 ul RP (GFP) 0.75 ul Template 2 ul ddH20 9 ul Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 98 C for 20 seconds 3. Annealing: 62 C for 15 seconds 4. Extension: 72 degrees, 15 sec 5. Repeat step 2-4 for 25 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | |||
| Gel Electrophoresis | David | Gel: 1) Ladder, 2) J48200 Banana Odor MPP, 3) J33204 XyIE + RBS T1, 4) J33204 XyIE + RBS T2, 5) fliC1 no ext C, 6) fliC1 No Ext 1, 7) fliC1 no ext 2, 8) flic1 no ext 3, 9) ladder,10) PP mCer 1, 11) DP mCer1 X+S, 12) PP mCit 1, 13) DP mCit 1 X+S, 14) PP NmCit 1, 15) DP Nmcit 1 X+S | |||
| PCR Overlap Extension | Victor | sprFLiC1 + GFP FliC2 (5 trials) | Hotstart PCR Protocol: Hotstart 12.5 ul FP (fliC1) 0.75 ul RP (GFP) 0.75 ul Template 2 ul ddH20 7 ul Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 98 C for 20 seconds 3. Annealing: 62 C for 15 seconds 4. Extension: 72 degrees, 2 min 5. Repeat step 2-4 for 25 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | ||
| Gel Extraction | Victor | Gel Extracion 1) Ladder, 2-7) spr GFP ff C, T1-T5 | |||
| Gel | Andrew | Gel 1) 1 KB+ Ladder, 2-4) pfliC C-T1-T2 | |||
| PCR | C-split mCer, C-split mCit, N-split mCer, ECFP | Hotstart PCR Protocol: Hotstart 12.5 ul FP (fliC1) 0.75 ul RP (GFP) 0.75 ul Template 2 ul ddH20 7 ul Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 98 C for 20 seconds 3. Annealing: 64 for 15 seconds 4. Extension: 72 degrees, 5 min 5. Repeat step 2-4 for 25 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | |||
| Liquid Cultures | Kevin | 1C3+FF GFP (Control, 1:3, 1:1, T1A, CB) | |||
| Gel | Beini | Gel: 1) 1 KB+ Ladder, 2-5) Cm Cer C, T1-T3, 6) 1 KB+ Ladder, 7-9) CmCit 1-3, 10) ECFP C, 11) 1 KB+, 12-15) NmCer C-3, 16) 1 KB+, 17-20) ECFP C, T1-T3 | |||
| Aug-04 | Liquid Cultures | Beini | pSB1C3 + pfliC GFP T3, BBa_1I3601, BBa_E0430, pSB1C3 + pfliC GFP T1 (1:1), pSB1C3 + pfliC GFP control, pSB1C3+ pflic T3, BBa_I719015, BBa_I13602 | ||
| PCR | Kevin | sprfliC1 extending on to GFP fliC2 and on to GFp | Hotstart PCR Protocol: Hotstart 12.5 ul FP (fliC1) 0.75 ul RP (GFP) 0.75 ul Template 3 ul ddH20 6ul Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 98 C for 20 seconds 3. Annealing: 62 for 15 seconds 4. Extension: 72 degrees, 2 min 5. Repeat step 2-4 for 25 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | ||
| Miniprep | Faisal, Beini | Bio-timer, IC3 FF GFP 1:1, pfliC GFP FliC2 IT3 T2B, 1T3 FFGFP T1A 100 ul, IT3 FF GFP 500 ul CB pfliC GFP fliC2 IT3 T1B, pfliC GFP fliC2 IT3 Ca, 1C3 ffGFP Trial 2 1:3 500 ul, pfliC GFP fliC2 IT3 T3A, 1C3 ffGFP Control 500 ul, apple scent | |||
| Digestions and Ligations | MPPs w/ NOt1 | Used the standard Fermentas Fast Digest Protocol | |||
| Gel | Gel: 1) 1 KB+ 2-4) spFF GFP 1-3, 5-8) spfliC GFP 1-3, Control | ||||
| Gel | Beini | Gel: 1)Ladder, 2) spFF GFP C, 3) MPP Bio-timer, 4) DIG biotimer fluorescent, 5) MPP 1C3 FFGFP 1:1, 6) DIG 1C3 FFGFP 1:1, 7) MPP pfliC GEP FliC2 IT3 T2B, 8) DIG pfliC GFP fliC2 IT3 T2B, 9) 1 KB+, 10) MPP It3 FFGFP T1A, 11) DIG IT3 FFgFP 1TA, 12) MPP biotimer, 13) Dig biotimer, 14) MPP IT3 FFgFP, 15) DIG IT3 FFGFP, 16) Ladder, 17) MPP pfliC GFP FliC2 IT3 T1B, 18)DIG pfliC GFP FLiC2 ITe T1B, 19) MPP pfliC GFP fliC2 IT3 Ca, 20) DIG pfliC GFP fliC2 IT3 Ca, 21) MPP 1C3 Ff GFP T2 1:3, 22) DIg 1C3 ffgfp T2 1:3, 23) 1 KB+ Ladder, 24) MPP pfliC GFP FliC2 IT3 T3A, 25) DIG pfliC GFP fliC2 1T3 T3A, 26) MPP 1C3 FFGFP Ctrl, 27) DIG 1C3 FFGFP Ctrl, 28) MPP apple scent,29) DIG apple scent, 30) Blank | |||
| Aug-05 | PCR | Kevin | fliC1 w/ added etra base pairs so we can digest w/ EcoRI | Hotstart PCR Protocol: Hotstart 12.5 ul FP (fliC1) 0.75 ul RP (GFP) 0.75 ul Template 2 ul ddH20 7 ul Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 98 C for 20 seconds 3. Annealing: 59 for 15 seconds 4. Extension: 72 degrees, 20 sec 5. Repeat step 2-4 for 25 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | |
| Gel | Gel: 1) 1 KB + Ladder, 2-5) spFF GFP 1-3,C, 6-7) spr fliC GFP GE 1-2 | ||||
| Rehydration | ECFP | ||||
| Heat Shock Transformation | EE0422 | ||||
| PCR | E0422 | Hotstart PCR Protocol: Hotstart 12.5 ul FP (fliC1) 0.75 ul RP (GFP) 0.75 ul Template 2 ul ddH20 7 ul Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 98 C for 20 seconds 3. Annealing: 54 for 15 seconds 4. Extension: 72 degrees, 30 sec 5. Repeat step 2-4 for 25 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | |||
| Miniprep | pSB1C3 + pfliC GFP (4 trials), BBa_I13601, BBa_I13602, BBa_I719015, BBa_E0430 | ||||
| Extractions | |||||
| Gel | Gel 1) Ladder, 2-5) spFFGFP 1-3, C, 6-8) ECFP 1-2, C | ||||
| Digestion | MPP pfliC GFP fliC2 IT3 T1B | Used standard NEB protocol with Eco R1 (0.5) | |||
| Digestion | Beini | MPPs w/ XbaI | Fast Digest Protocol: 2 ul Template 2 ul 10x FD Buffer Green 1 ul Xbai 15 ul H20 | ||
| PCR | spr fliC+GFP | Hotstart PCR Protocol: Hotstart 12.5 ul FP (fliC1) 0.75 ul RP (GFP) 0.75 ul Template 2 ul ddH20 7 ul Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 98 C for 20 seconds 3. Annealing: 59 for 15 seconds 4. Extension: 72 degrees, 2 min 5. Repeat step 2-4 for 25 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | |||
| Gel on bottom | |||||
| Gel on Top | |||||
| PCR | MPP pfliC GFP fliC2 IT3 T1B as template | Hotstart PCR Protocol: Hotstart 12.5 ul FP (fliC1) 0.75 ul RP (GFP) 0.75 ul Template 2 ul ddH20 7 ul Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 98 C for 20 seconds 3. Annealing: 59 for 15 seconds 4. Extension: 72 degrees, 2.5 min 5. Repeat step 2-4 for 25 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | |||
| Gel | Beini | Gel 1) Ladder,2-3) spF GFP 1-2, 4) DP mCer 1 XS, 5) DP mCit 1 XS, 6) DP NmCit 1 XS | |||
| PCR Purifications | sfliC | ||||
| Liquid Culture | ECFP | ||||
| Gel Extraction | spF GFP land 2 |
Given that the vast majority of our work now is in the lab, we will only be updating the "Labwork" section of the Notebook.
