Team:Queens Canada/Notebook/Week14

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Notebook - Week 14

DateProtocolPeopleDNA (if relevant)Quantities and Parameters (if relevant)Notes on Protocol/ Gel Lanes
Jul-27Gel   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.
 PCRPhillippFliC 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 PhillippFLiC 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 CulturesBeiniXLI Blue [Tet]  
 Motility Plate BeiniXLI Blue [Tet]  
 Heat Shock TransformationBeinifliC C2, MPP T  
 Gel ElectrophoresisPhilip  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 ExtractionDavidF2 GFP PP T4. F2 GFP PP T1, pF GE2 GFP 1 T1, pf GE2 GFP2 T1  
 PCRFaisal, DavidXyIEHotstart 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 2Hotstart 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 PlatesBeini   
Jul-28Motility Plate CheckKevinGFP  
 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-29PCR Overlap ExtensionDavid, KevinAdding promotor to RBS fliCHotstart 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
 MiniprepDavidMPP 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-30PCR ExtensionDavidRBS 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  
 DigestionVictorpFliC 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
 PCRVictor AndrewPCR 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  
 GelFaisal  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 XyLEHotstart 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 PromotorHotstart 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
 
 GelAndrew, 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 ExtractionAndrewpFLiC GFP, fliC  
Jul-31Mobility Plates XLI Blue, BL21, XLI Blue Top 10  
 PCRDavid, VictorpFliC GFP 1-4, CHotstart 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
 
 DigestionDavidDIG 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 CHotstart 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
 GelFaisal  Gel:
1) Ladder, 2) pFLiC Suffix C, 3-7) pFliC GFP 1-4, C
 PCR Overlap ExtensionVictorpfliC added Suffix 1-4, CHotstart 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 ExtractionBeinipfliC 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 PurificationVictorpfliC Suffix 1-4  
 DigestionFaisalFliC T1--> S+P, XyIE ---> X+PUsed standard Fast Digested Protocol 
      
August-1stPCRVictor, DavidpFliC+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
 
 LigationFaisalUsing 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 PlatesVictorMade 10  
 Gel   Gel:
1) 1 KB+ Ladder, 2-6) re pfliC GFP 1-4, 6-8) adsuf pfliC GFP C, 1-2.
 Gel ExtractionAndrewre 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 PSB1T3NEB 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 ---- FliC2Hotstart 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-02PCRVictor, DavidTwo 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  
 GelFaisal  Gel:
1) KB+ ,2-5)LtG ff GFP 1T3 Control -T3, 6-8) pfGFP F2 1-3
 PCRDavidXyIEHotstart 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 extractionVictorasuf IDT ffGFP 1-4, and asuf pFF GFP 1-3  
 Gel ExtractionBeinipF GFP F2 T1-T3  
 Gel Extraction Beiniadsuf 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 + TransformationsFaisalplasmid backbone pSb1C3.  
 Liquid Cultures J33204, XyIE + RBS and J48200, Banana Odor  
 Liquid CulturesDavidLIG pflic GFP fliC 2 + pSBIT3, Ca, T3A, T2B and T1B, apple scent K395602, and bio- timer K088006  
Aug-03MiniprepVictorJ33204 T1-T2, J48200 Banana odor  
 PCRVictorFliC1 No extensions ( Templates pfliC GE, LP: fliC1 LP, Rp : fliC RPHotstart 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 PurificationsDavidmCer1, mCer2, mCit1, mCit2, Nmcit1, Nmcit 2 all PCR Products from 06/22  
 Digestion of PCR purification productsDavidPP 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 TransformationsFaisalBBa_ I13602, I719015, I13601, E0430  
 PCR pFliC 1 with added base pairs (2 trialsHotstart 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 ElectrophoresisDavid  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 ExtensionVictorsprFLiC1 + 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 ExtractionVictor  Gel Extracion
1) Ladder, 2-7) spr GFP ff C, T1-T5
 GelAndrew  Gel
1) 1 KB+ Ladder, 2-4) pfliC C-T1-T2
 PCR C-split mCer, C-split mCit, N-split mCer, ECFPHotstart 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 CulturesKevin1C3+FF GFP (Control, 1:3, 1:1, T1A, CB)  
 GelBeini  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-04Liquid CulturesBeinipSB1C3 + pfliC GFP T3, BBa_1I3601, BBa_E0430, pSB1C3 + pfliC GFP T1
(1:1), pSB1C3 + pfliC GFP control, pSB1C3+ pflic T3, BBa_I719015, BBa_I13602
  
 PCRKevinsprfliC1 extending on to GFP fliC2 and on to GFpHotstart 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
 
 MiniprepFaisal, BeiniBio-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/ NOt1Used the standard Fermentas Fast Digest Protocol 
 Gel   Gel:
1) 1 KB+ 2-4) spFF GFP 1-3, 5-8) spfliC GFP 1-3, Control
 GelBeini  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-05PCRKevinfliC1 w/ added etra base pairs so we can digest w/ EcoRIHotstart 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 E0422Hotstart 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 T1BUsed standard NEB protocol with Eco R1 (0.5) 
 DigestionBeiniMPPs w/ XbaIFast Digest Protocol:
2 ul Template
2 ul 10x FD Buffer Green
1 ul Xbai
15 ul H20
 
 PCR spr fliC+GFPHotstart 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 templateHotstart 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
 
 GelBeini  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.