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Team:Queens Canada/Notebook/Week12
From 2012.igem.org
Notebook - Week 12
| Date | Protocol | People | DNA (if relevant) | Quantities and Parameters (if relevant) | Notes on Protocol/ Gel Lanes |
|---|---|---|---|---|---|
| Jul-16 | Fast Digestion | Beini, Faisal | Fast Digest: 1) 2ul [GFP + pSB1C3] + 17 ul MM + 1 ul Not1 2) 2ul [SmtA + fliC C1 + pSB1C3 T1] + 17 ul MM. + 1 ul Not1 3) 2ul [SmtA + fliC C1 + pSB1C3 T2] + 17 ul MM + 1 ul Not1 4) 2ul [FliC C2 T1] + 17 ul MM + 1 ul Not1 5) 2ul [FliC C2 T2] + 17 ul MM + 1 ul Not1 6) 2ul [FliC C2 + pSB1A3 T2 (2) ] + 17 ul MM + 1 ul Not1 7) 2ul [Strong RBS + fliC C1 T2] + 17 ul MM. + 1 ul Not1 8) 2ul [Med RBS + fliC C1 T2] +17 ul MM + 1 ul Not1 9) 2ul [fliC C1 + pSB1A3 T2] + 17 ul MM + 1 ul Not1 10) 2ul [ fliC C2 T1] + 17 ul MM + 1 ul Not1 | Fast Digestion Mastermix (10 trials): Water 15 ul = 150 ul 10x FD Green Buffer 2 ul = 20 ul + Reagents as listed in cell to the left | Gel (All are Not1 digests) 1) Ladder 1 KB+, 2) GFP + pSB1C3, 3) SmTA + fliC C1 + pSB1C3 T1, 4) SmtA + fliC C1 + pSB1C3 T2, 5) fliC C2 T1, 6) fliC C2 T2, 7) Ladder 1 KB+, 8) fliC C2 + pSB1A3 T2 (2), 9) Strong RBS + fliC C1 T1, 10) Med RBS + fliC C1 T2, 11) fliC C1 + pSB1A3 T2, 12) fliC C2 T1 heat denaturing |
| PCR | Andrew, Phillip | PCR for ( 2 trials, 1 control) mgfp ( BBa_K197018), ech (vanillin) (BBa_K238028), PBrR (MPP trial 1 & 2) (BBa_I721002), ECEP (BBa_E0022), xyiE (BBa_K147002), N-split mCer ( BBa_K323011), C-split mCer (BBa_K323012), fcs (BBa_K238029), streptavidin ( BBa_K283010), Csplit mCitr (BBa_K323055) | 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 Total: 50 ul Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 95 C for 20 seconds 3. Annealing: 50 C 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 | Gel (All NotI Digests)/Actual Bands 1) 1 KB+ Ladder 2) GFP (E0040) + pSB1C3/ ~1500 3) SmtA+fliC1+pSB1C3 T1 / ~2200 4) SmtA+fliC1+pSB1C3 T2/~ 2200 5) fliC C2 (pCR2.1 (synthesis plasmid)) T1/ 700, 4-5000 6)fliC C2 (pCR2.1) T2/ ~2100, ~1800 7) Ladder/ ~1400 8) fliC C2 + pSB1A3/ ~1400 9) Strong RBS fliC C1 T1/ ~2000 10) Med RBS fliC C1 T2/ ~2000 11) fliC C1 + pSB1A3 T2/ ~ 1400 12) fliC C1 T1 Heat Denatured/ ~5000 | |
| PCR Gel | Phillip, Andrew | Small Gel: 1) Ladder 1 KB+, 2) ech C, 3) ech 1, 4) ech 2, 5) ECFP C, 6) ECFP 1, 7) ECFP 2 | Large Gel Note ( lane order is reversed) 1) Ladder, 2) xyIE C, 3) xyIE 1, 4) xyIE 2, 5) PBrR1 C (written as mgfPC), 6) PBrR1 1 (Written as mgfP1), 7) pBrR1 2 (written as mgfp2), 8) Ladder, 9) pBrR2 C, 10) PBrR2 1, 11)PBrR2 2, 12) Csplit mCitr C, 13) Csplit mCitr 1, 14) Cplit mCitr 2. | Large Gel Note 1) Ladder, 2) N-split mCer C, 3) N-split mCer 1, 4) N-split mCer 2, 5) C-split mCer C, 6) C-split mCer 1, 7) C-split mCer 2, 8) Ladder, 9) fcs C, 10) fcs 1, 11)fcs 2, 12) Strep C, 13) Strep 1, 14) Strep 2. | |
| Jul-17 | PCR | PCR of fliC1 using promoter primer | |||
| Rehydration | BBa_B0034 (RBS) (2 trials) | Thermopol Protocol: (5 trials) Thermopol: 5 ul dNTPs: 1 ul LP: 1.5 ul RP: 1.5 ul DNA Template: 10 ul Vent Pol: 0.7 ul MgSO4: 2 ul H2O: 38.3 ul Total: 50 ul Program:Not recorded | Made another tube of 5x XLI Blue DNA using 10 ul 10X XLI Blue DNA and 40 ul TE included LP & RP in mastermix | ||
| Heat Shock Transformation | Andrew | RBS B0034 | |||
| Jul-18 | Bacterial Cell Culture | Phillip | RBS B0034 T1, T2 | Took bottle of 2xTY from Chin Sang's lab + 1 box of yellow pipette tips | |
| Gel | Dan, Beini | Gel: 1)Ladder, 2)pFliC C, 3) pFliC 1, 4) pFliC 2, 5) pFliC 3, 6) pfliC 4. | |||
| Fast Digestion/ Gel | Phillip, Beini | fliC1 w/ SpeI (six trials) | Fast Digest Protocol: 2 ul DNA fiC1 2 ul 10x FD Buffer Green 1 ul SpeI (FD) 15 ul H20 | Gel: 1) Ladder, 2)fliC1 T1 [1], 3) fliC1 T1 [2], 4) fliC1 T2 [1], 5) fliC1 T2 [2], 6) fliC1 T3 [1], 7) fliC1 T3 [2] | |
| Glycerol Stock | Kevin | Used the Glycerol stock protocol. | |||
| Fast Digest | PCR Product of: GFP, smTa, Gold Binding Protein (GoIB) | Fast Digest Protocol: 2 ul DNA fiC1 2 ul 10x FD Buffer Green 1 ul SpeI (FD) 15 ul H20 | Done in preparation of PCR overlap extension | ||
| PCR Overlap extension | Beini, Faisal | fliCC1 and GFP, smTa, Gold Binding Protein (GoIB) | Thermopol Protocol: Thermopol: 5 ul dNTPs: 1 ul LP pfliC1: 1.5 ul RP insert: 1.5 ul pfliC1 template DNA: 2.5 ul insert Template DNA: 2.5 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: 98 C for 20 seconds 3. Annealing: 65 C for 15 seconds 4. Extension: 72 degrees, 60 seconds 5. Repeat step 2-4 for 25 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | ||
| PCR | Phillip, Andrew | fliC (four trials one control) | Thermopol Protocol: Thermopol: 5 ul dNTPs: 1 ul LP pfliC1: 1.5 ul RP insert: 1.5 ul Template DNA: 10 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: 98 C for 20 seconds 3. Annealing: 54 C for 17 seconds 4. Extension: 72 degrees, 60 seconds 5. Repeat step 2-4 for 36 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | ||
| Gel | Kevin | Gel: 1)1KB+, 2) pFliC C, 3)pFliC 1, 4) pFliC 2, 5) pFliC 3 6)pFliC 4. 7) pFliC1 + GFP C, 8) pFliC 1+ GFP 1, 9)1 KB+, 10) pFliC 1+GFP 2, 11) pFliC 1 + GolBC, 12) pFliC 1 + GoIB1, 13) pFliC 1 + GoIB 2, 14) pFliC 1 + Smt C, 15) pFliC 1 + Smt1 | |||
| Jul-19 | Liquid Cultures | Kevin | Liquid Culture of DH5α from last night | ||
| PCR Overlap Extension | Faisal | PCR Overlap Extension from yesterday with more plymerase and dNTPs being put in the same PCR tubes. The tubes being done again are: 1) pFliC1 + GFP C, 2) pFliC 1+ GFP 1, 3)1 KB+, 4) pFliC 1+GFP 2, 5) pFliC 1 + GolBC, 6) pFliC 1 + GoIB1, 7) pFliC 1 + GoIB 2 | Thermopol Protocol: Thermopol: 5 ul dNTPs: 1 ul LP pfliC1: 1.5 ul RP insert: 1.5 ul pfliC1 template DNA: 2.5 ul insert Template DNA: 2.5 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: 98 C for 20 seconds 3. Annealing: 68 C for 15 seconds 4. Extension: 90 degrees, 60 seconds 5. Repeat step 2-4 for 15 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | ||
| Digestion | Beini, Phillip | GFP digestion w/ X and S | Standard NEB Protocol: Used 2 ul of GFP1 PCR product found in bundle w/ GFP 1,2,4. | ||
| Miniprep | Kevin | RBS B0034 | |||
| PCR Overlap Extension | Beini, Phillip | Thermopol Protocol: Thermopol: 2.5 ul dNTPs: 0.5 ul GFP Digest w/ XS: 5 ul fliC2 Digest w/ XS: 5 ul pfliC1 PCR product: 5 ul Vent Pol: 0.35 ul MgSO4: 1 ul H2O: 5.64 ul Total: 25 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: 60 C for 15 seconds 4. Extension: 72 degrees, 30 seconds 5. Repeat step 2-4 for 15 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | |||
| Plating | Kevin | Top 10 using sealed competent cells, | |||
| Motility Assay Test | Kevin | Innoculated the center of two motility plates using two colonies from the DH5α culture plate which were prewarmed for five minutes. They were innoculated by stabbing colony and then stabbing center of plate | |||
| Gel on PCR Overlap Extension | Faisal | Gel: 1) Ladder, 2) pFliC1 + GFP C, 3) pFliC 1+ GFP 1, 4)1 KB+, 5) pFliC 1+GFP 2, 6) pFliC 1 + GolBC, 7) pFliC 1 + GoIB1, 8) Ladder, 8) pFliC 1 + GoIB2, 9) pfliC 1 + fliC2 + GFP C, 10) pfliC 1+ fliC 2 + GFP 1, 11)pfliC1+ fliC 2 + GFP 2, 12) pfliC 1 + fliC2 + GFP 3 | |||
| Jul-20 | Glycerol Stock | Andrew | DH5α | ||
| Liquid Cultures | Beini | TOP10 | |||
| PCR Overlap Extension | Phillip Andrew | Thermopol Protocol: Thermopol: 5 ul dNTPs: 1 ul Template: 10 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: 98 C for 20 seconds 3. Annealing: 58 C for 20 seconds 4. Extension: 72 degrees, 45 seconds 5. Repeat step 2-4 for 15 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | |||
| Motility Assay Test | Beini | TOP 10 (Four colonies each) | |||
| Innoculation | BL21 and XLI Blue using glycerol stocks with antibiotic | ||||
| Gel Electrophoresis | Phillip, Faisal | Gel: 1) Blank, 2) Ladder, 3) pfliC1 + GFP 1, 4) pfliC1 + GFP 2, 5) pfliC1 + GoIB1, 6) pfliC1 + GoIB2, 7) Ladder, Rest is blank | |||
| Gel Extraction | Refer to Gel Extraction protocol | ||||
| Jul-21 | Motility Plate Results | TOP 10 showed no motility. BL21 plate turned out fine | |||
| Motility Assay of BL21 | Beini | BL21 | |||
| PCR of Gel Extractions | Beini | pfliC1 + GFP, pfliC1 + GoIB | Thermopol Protocol: Thermopol: 5 ul dNTPs: 1 ul LP pfliC1: 1.5 ul RP insert: 1.5 ul DNA Template: 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: 98 C for 20 seconds 3. Annealing: 60 C for 15 seconds 4. Extension: 72 degrees, 30 seconds 5. Repeat step 2-4 for 15 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | Gel: 1) Ladder 1KB+, 2) pFliC1 + GFP C, 3) pFliC 1+ GFP 1, 4) pFliC 1+GFP 2, 5) pFliC 1 + GolBC, 6) pFliC 1 + GoIB1, 7) pFliC 1 + GoIB 2, 8)Blank | |
| Jul-22 | Motiliy Assay | XLI Blue | |||
| PCR | Andrew | Protocol not listed Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 95 C for 20 seconds 3. Annealing: 60 C for 15 seconds 4. Extension: 72 degrees, 1 minute 5. Repeat step 2-4 for 15 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | |||
| Glycerol Stock | Faisal | TOP 10 Liquid Culture | |||
| Gel | Gel of round two PCR of gel extractions | Gel: 1) Ladder 1KB+, 2) pFliC1 + GFP C, 3) pFliC 1+ GFP 1, 4) pFliC 1+GFP 2, 5) pFliC 1 + GolBC, 6) pFliC 1 + GoIB1, 7) pFliC 1 + GoIB 2, 8)Blank | |||
| PCR | Beini, Andrew | pFliC1 + GFP (XS) + fliC2 (XS) | Used Vent Protocol: Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 95 C for 20 seconds 3. Annealing: 55 C for 30 seconds 4. Extension: 60 degrees, 1 minute 5. Repeat step 2-4 for 20 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour | Gel: 1) Ladder, 2) pfliC1 + fliC2 +GFP C, 3)pfliC1 + fliC2 +GFP 1, 4)pfliC1 + fliC2 +GFP 2, 5)pfliC1 + fliC2 +GFP 2, 6) pfliC1 + SmtA C, 7) pfliC1 + SmtA 1, 8) pfliC1 + SmtA 2 | |
| Motility Assay | XLI Blue | ||||
| PCR | Phillip | pFLIC 1 +fliC2 + GFP (6 trials) and pFliC1 + fliC2 + GFP Control (3 rials | Thermopol Protocol: Thermopol: 5 ul dNTPs: 1 ul LP pfliC1: 1.5 ul RP insert: 1.5 ul DNA Template: 10 ul Vent Pol: 0.7 ul MgSO4: 1 ul H2O: 28.3 ul Total: 28.3 ul Using LP of fliC C1 and RP of respective part. 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, 120 seconds 5. Repeat step 2-4 for 15 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour |
Monday July 16
To start the week off, Kevin went over some vital guidelines for lab work that we need to follow in order for us to keep organized and avoid wasting time/reagents. Then, since there were just the five of us, we all went into the lab to work on various things. Beini and Faisal did a FastDigest on 9 miniprep products to see if our ligations worked. They digested for 35 minutes instead of the usual 30, in hopes of better results. According to the gel, all the ligations failed. Andrew and Phillip performed a massive PCR of BioBrick parts we haven’t successfully PCR’d yet. Kevin did some reorganization of labwork, such as analyzing previous gels to check for digestion success.
Tuesday July 17th
This morning, Kevin and Andrew met with Dr. Smith to discuss gene sequences for cohesins and dockerins. Phillip and Andrew continued the gel analysis that Kevin started yesterday. Kevin, Faisal, and Beini worked on making primers for our next order. We planned to put in a rush order for only the primers that we would need to make a working GFP construct. In order for the primers to arrive tomorrow, we had to get our order in by 3 pm, so when everyone came back from lunch at 2 pm, we put all our heads together to grind out those primers. However, at 2:52 pm we ran into some unforeseen issues that required some consideration, so then we decided to chill out and just put an order in for 3 pm tomorrow. In the lab, we rehydrated a RBS BioBrick part and transformed it, liquid cultured some BL21 and XL1 Blue, and added a promoter to fliC1 using PCR.
Wednesday July 18th
Today, we put in our rush order for primers so that they hopefully arrive tomorrow. Kevin and Maggie met with a representative from KEDCO (Kingston Economic Development Corporation) to ask for advice on how to garner support for SynthetiQ from the local community (eg. educational institutions, dancers/choreographers). Later in the day, they also met with Dr. Allingham to discuss molecular modelling with a software called Coot, that we might use for modelling our chimeric flagellin. As for labwork, we made liquid cultures of the RBS from yesterday, made glycerol stocks of XL1 Blue and BL21, and digested fliC1 with SpeI to confirm the presence of that cut site. We also attempted a PCR overlap extension of pfliC1 with three different inserts (GFP, smtA, golB).
Thursday July 19th
Our rush order for primers was supposed to arrive by 5 pm, so we kept our eyes peeled for that the entire day, but alas they did not arrive by that time. For labwork, we re-ran yesterday’s PCR overlap extension and added more dNTPs and polymerase, because from the gel it looked like a lot of primers didn’t bind in the original PCR. We also tried PCR overlap extension using pfliC1 and X/S digests of fliC2 and GFP, with no primers. We attempted our first motility assay by stabbing a colony of DH5-alpha onto a soft agar plate. In the afternoon, we worked on primers for conversion of parts to BB-2 standard, and for parts we didn’t have primers for previously. Today was also Kevin’s birthday! Happy birthday Kevin.
Friday July 20th
For our motility assay of DH5-alpha, we got a nicely shaped circle ~1.1 cm. However, DH5-alpha is not supposed to express native flagella, so we will have to look further into this. For our next motility assay of TOP10, we decided to try one soft agar plate and one LB plate. We also divided the plates into quarters and tried four different levels of stabbing (i.e. from shallow to deep). For overlap PCR extension, we ran another round of PCR for pfliC1 + fliC2 + GFP, this time just adding a bit more polymerase and not dNTPs (because they probably didn’t get used up). Then we ran a gel of all the PCR overlap extensions we have done so far. In the afternoon, Kevin showed us how to do a gel extraction, and cut out bands of fliC1 + GFP and fliC1 + GolB from the aforementioned gel. For the rest of the day, Phillip did some filming for our project overview video as we took turns speaking for the camcorder.
