Team:TU Darmstadt/labbook

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Labbook | Protocols | Materials |
13.08 gBLOCK assembly of CMK and TLO
  ·       PCR mixture
  ·       1 µL of each gBLOCK
  ·       CMK: B_01 – B_04
  ·       TLO: A_01 – A_06
  ·       10 µL 5x Q5 Reaction Buffer
  ·       2 µL dNTPs
  ·       1 µL Q5 Hot Start Polymerase
  ·       10 µL 5x Q5 High GC Enhancer
  ·       1 µL primer suffix-R (10 mM)
  ·       1 µL primer prefix_R (10 mM)
   
  ·       PCR program (40 cycles)
  ·       initial denaturation 94°C, 100s
  ·       denaturation 94°C, 55s
  ·       annealing 64°C, 55s
  ·       elongation 72°C, 120s
  ·       final elongation 72°C, 300s
   
  ·       preparative 1% agarose gel
  ·     gel displays bands of expected size à assembly was successful
   
   
13.08 Purification
  ·       using Wizard SV Gel and PCR Clean-Up System (Promega)
  ·       TLO c = 34,4 ng/µL
  ·       CMK c = 35,6 ng/µL
   
19.08 Overlap extension PCR
  ·      
  reaction mixture (50 µL total volume)
  ·     25 µL Q5 High Fidelity 2x Master Mix (NEB)
  ·     1 µL template
  ·     1 µL forward primer (10 mM)
  ·     1 µL reverse primer (10 mM)
  ·     22 µL nuclease-free H2O
   
  ·       template and primer specifications
  ·     pBAD1: pSB1C3[BBa_K206000] + frag1_for + frag4_rev
  ·     CMK: CMK gBLOCK assembly + frag2_for + frag2_rev
  ·     terminator: pSB1AK3[BBa_B0014] + frag3_for + frag3_rev
  ·     pBAD4: pSB1C3[BBa_K206000] + frag4_for + frag4_rev
  ·     TLO: TLO gBLOCK assembly + frag5_for + frag5_rev
  ·     pSB1C3: pSB1C3[fsC] + vector_for + vector_rev
   
  ·       PCR program (35 cycles)
  ·     initial denaturation: 98°C, 60s
  ·     denaturation: 98°C, 45s
  ·     annealing: 60°C, 40s
  ·     elongation: 72°C, 90s
  ·     final elongation: 72°C, 300s
   
  ·       preparative 1% agarose gel
  ·     gel displays byproducts
  à subsequent purification and amplification of desired bands (framed)
   
19.08 Purification
  ·       using Wizard SV Gel and PCR Clean-Up System (Promega)
  ·       pBAD1 c = 10 ng/µL
  ·       CMK c = 30 ng/µL
  ·       terminator c = 7 ng/µL
  ·       pBAD4 c = 11 ng/µL
  ·       TLO c = 22 ng/µL
  ·       pSB1C3 c = 16 ng/µL
   
19.08 Amplification PCR of pBAD1, terminator and pBAD4
  ·       reaction mixture (25 µL total volume)
  ·     12,5 µL Q5 High Fidelity 2x Master Mix (NEB)
  ·     1 µL template
  ·     1 µL forward primer (10 mM)
  ·     1 µL reverse primer (10 mM)
  ·     9,5 µL nuclease-free H2O
   
  ·       template and primer specifications
  ·     pBAD1: pBAD1 (c = 10 ng/µL, 19.08) + frag1_for + frag4_rev
  ·     terminator: terminator (c = 7 ng/µL, 19.08) + frag3_for + frag3_rev
  ·     pBAD4: pBAD4 (c = 11 ng/µL, 19.08) + frag4_for + frag4_rev
   
  ·       PCR program (35 cycles)
  ·      initial denaturation: 98°C, 60s
  ·      denaturation: 98°C, 45s
  ·      annealing: 70°C, 30s
  ·      elongation: 72°C, 30s
  ·      final elongation: 72°C, 300s
   
  ·       analytical 1% agarose gel
  ·       pBAD1 shows single band of expected size, 70°C is the optimal annealing temperature
  ·       terminator and pBAD4 show no bands
   
19.08 Gradient PCR of the amplification of pBAD1, terminator and pBAD4
  ·       reaction mixture (25 µL total volume)
  ·     12,5 µL Q5 High Fidelity 2x Master Mix (NEB)
  ·     4 µL template
  ·     1 µL forward primer (10 mM)
  ·     1 µL reverse primer (10 mM)
  ·     6,5 µL nuclease-free H2O
   
  ·       template and primer specifications
  ·     pBAD1: pBAD1 (c = 10 ng/µL, 19.08) + frag1_for + frag4_rev
  ·     terminator: terminator (c = 7 ng/µL, 19.08) + frag3_for + frag3_rev
  ·     pBAD4: pBAD4 (c = 11 ng/µL, 19.08) + frag4_for + frag4_rev
   
  ·       PCR program (35 cycles)
  ·       initial denaturation 98°C, 60s
  ·       denaturation 98°C, 45s
  ·       annealing 55°C/57°C/61°C/65°C, 30s
  ·       elongation 72°C, 30s
  ·       final elongation 72°C, 300s
   
  ·       analytical 1% agarose gel
  ·       optimal annealing temperature for terminator is 55°C
  ·       optimal annealing temperature for pBAD4 is 55°C
   
   
20.08 Purification of PCR batches pBAD1 (Amplification PCR, 19.08), terminator 55°C (Gradient PCR, 19.08) and pBAD4 55°C (Gradient PCR, 19.08)
  ·       using Wizard SV Gel and PCR Clean-Up System (Promega)
  ·       pBAD1 c = 45 ng/µL
  ·       terminator c = 44 ng/µL 260/280 = 1,67 260/230 = 0,73
  ·       pBAD4 c = 57,7 ng/µL 260/280 = 1,81 260/230 = 0,67
   
21.08 Amplification PCR of CMK, TLO and pSB1C3
  ·       PCR mixture (50 µL total volume)
  ·       25 µL Q5 High Fidelity 2x Master Mix
  ·       1 µL template
  ·       1 µL forward primer
  ·       1 µL reverse primer
  ·       22 µL nuclease-free H2O
   
  ·       template and primer specifications
  ·     CMK: CMK (c = 30 ng/µL, 19.08) + frag2_for + frag2_rev
  ·     TLO: TLO (c = 22 ng/µL, 19.08) + frag5_for + frag5_rev
  ·     pSB1C3: pSB1C3 (c = 16 ng/µL, 19.08) + vector_for + vector_rev
   
  ·       PCR program (35 cycles)
  ·       initial denaturation 98°C, 60s
  ·       denaturation 98°C, 45s
  ·       annealing 55°C, 40s
  ·       elongation 72°C, 90s
  ·       final elongation 72°C, 300s
   
  ·       analytical 1% agarose gel
  ·       gel displays critical amount of byproducts
   
21.08 Gradient PCR of the amplification of CMK, TLO and pSB1C3
  ·       Amplification using Q5 Polymerase
   
  ·       PCR mixture (50 µL total volume)
  ·       25 µL Q5 High Fidelity 2xMaster Mix
  ·       2 µL template
  ·       1 µL forward primer (10 mM)
  ·       1 µL reverse primer (10 mM)
  ·       22 µL nuclease-free H2O
   
  ·       PCR program (35 cycles)
  ·       initial denaturation 98°C, 60s
  ·       denaturation 98°C, 45s
  ·       annealing 55°C/56,7°C/60,4°C/62,9°C/64,9°C, 30s
  ·       elongation 72°C, 30s
  ·       final elongation 72°C, 300s
   
  ·       Amplification using Pfu Polymerase (homehold)
   
  ·       PCR mixture (50 µL total volume)
  • 2 µL template
  • 10 µL 5x Phusion-Buffer
  • 2 µL dNTPs
  • 2 µL DMSO
  • 4 µL Pfu-Polymerase
  • 2 µL MgCl2 (50 mM)
  • 1 µL forward primer (10 mM)
  • 1 µL reverse primer (10 mM)
  • 26 µL nuclease-free H2O
   
  ·       PCR program (30 cycles)
  • initial denaturation 98°C, 60s
  • denaturation 98°C, 30s
  • annealing 55°C/56,4°C/59,3°C/62,1°C/65°C, 30s
  • elongation 72°C, 720s
  • final elongation 72°C, 600s
   
  ·       template and primer specifications
  ·     CMK: CMK (c = 30 ng/µL, 19.08) + frag2_for + frag2_rev
  ·     TLO: TLO (c = 22 ng/µL, 19.08) + frag5_for + frag5_rev
  ·     pSB1C3: pSB1C3 (c = 16 ng/µL, 19.08) + vector_for + vector_rev
   
  ·       preparative 1% agarose gel
  ·     amplification with Pfu Polymerase yields lesser byproducts than amplification with Q5 polymerase
  ·     for all three templates higher annealing temperatures diminish the amount of by products
   
22.08 Purification of CMK, TLO and pSB1C3 from preparative gel
  ·       using Wizard SV Gel and PCR Clean-Up System (Promega)
  ·       CMK c = 154,2 ng/µL 260/280 = 1,92 260/230 = 1,88
  ·       TLO c = 37,9 ng/µL 260/280 = 1,90 260/230 = 0,60
  ·       pSB1C3 c = 120,8 ng/µL 260/280 = 1,89 260/230 = 1,64
   
23.08 Amplification of TLO
  ·       PCR mixture (50 µL total volume)
  • 2 µL TLO (c = 37,9 ng/µL, 22.08)
  • 10 µL 5x Phusion-Buffer
  • 2 µL dNTPs
  • 2 µL DMSO
  • 4 µL Pfu-Polymerase
  • 2 µL MgCl2 (50 mM)
  • 1 µL frag5_for (10 mM)
  • 1 µL frag5_rev (10 mM)
  • 26 µL nuclease-free H2O
   
  ·       PCR program (30 cycles)
  • initial denaturation 98°C, 60s
  • denaturation 98°C, 30s
  • annealing 55°C, 30s
  • elongation 72°C, 300s
  • final elongation 72°C, 600s
   
  ·       preparative 1% agarose gel
  ·     gel displays no distinctive bands
   
24.08 Amplification of TLO
  ·       PCR mixture (50 µL total volume)
  • 1 µL TLO (c = 37,9 ng/µL, 22.08)
  • 10 µL 5x Phusion-Buffer
  • 2 µL dNTPs
  • 2 µL DMSO
  • 4 µL Pfu-Polymerase
  • 2 µL MgCl2 (50 mM)
  • 1 µL frag5_for (10 mM)
  • 1 µL frag5_rev (10 mM)
  • 27 µL nuclease-free H2O
   
  ·       PCR program (30 cycles)
  • initial denaturation 98°C, 60s
  • denaturation 98°C, 30s
  • annealing 56°C, 30s
  • elongation 72°C, 720s
  • final elongation 72°C, 600s
   
  ·       preparative 1% agarose gel
  ·     gel displays no distinctive bands (just like on 23.08) à probably the template is poorly
   
24.08 Amplification of TLO
  ·       PCR mixture (50 µL total volume)
  • 1 µL TLO gBLOCK assembly (c = 34,4 ng/µL)
  • 10 µL 5x Phusion-Buffer
  • 2 µL dNTPs
  • 2 µL DMSO
  • 4 µL Pfu-Polymerase
  • 2 µL MgCl2 (50 mM)
  • 1 µL frag5_for (10 mM)
  • 1 µL frag5_rev (10 mM)
  • 27 µL nuclease-free H2O
   
  ·       PCR program (30 cycles)
  • initial denaturation 98°C, 60s
  • denaturation 98°C, 30s
  • annealing 56°C, 30s
  • elongation 72°C, 720s
  • final elongation 72°C, 600s
   
  ·       preparative 1% agarose gel
  ·     gel displays no distinctive bands
25.08 Amplification of TLO
  ·       PCR mixture (50 µL total volume)
  • 2 µL TLO gBLOCK assembly (c = 34,4 ng/µL)
  • 10 µL 5x Phusion-Buffer
  • 2 µL dNTPs
  • 2 µL DMSO
  • 4 µL Pfu-Polymerase
  • 2 µL MgCl2 (50 mM)
  • 1 µL frag5_for (10 mM)
  • 1 µL frag5_rev (10 mM)
  • 26 µL nuclease-free H2O
   
  ·       PCR program (30 cycles)
  • initial denaturation 98°C, 60s
  • denaturation 98°C, 30s
  • annealing 55°C, 30s
  • elongation 72°C, 300s
  • final elongation 72°C, 600s
   
  ·       preparative 1% agarose gel
  ·       gel displays the desired band of ~2500 bp as well as byproducts
   
25.08 Purification of TLO
  ·       using Wizard SV Gel and PCR Clean-Up System (Promega)
  ·       TLO c = 19 ng/µL
   
25.08 InFusion
  ·       reaction mixture (71 µL total volume)
  ·       1µl pBAD1 (130 bp, c = 45 ng/µL, 45 ng)
  ·       1µl pBAD4 (130 bp, c = 57,7 ng/µL, 58 ng)
  ·       1µl terminator (100 bp, c = 44 ng/µL, 44 ng)
  ·       42µl TLO (2440 bp, c = 19 ng/µL, 780 ng)
  ·       4,5µl CMK (1850 bp, c = 154,2 ng/µL, 680 ng)
  ·       3,5µl pSB1C3 (2100 bp, c = 120,8 ng/µL, 360 ng)
  ·       18µl 5x InFusion Pfu MasterMix
   
  ·       treatment of the reaction mixture according to InFusion protocol
   
  ·       transformation into E.coli Top10 according to heat shock protocol
  ·       no colonies grew on LB-Cam plates, most likely the reaction volume was to high
   
26.08 Amplification of TLO
  ·        PCR mixture (50 µL total volume)
  ·      
  1 µL TLO gBLOCK assembly (c = 34,4 ng/µL)
  ·       1 µL frag5_rev (10 mM)
  ·       1 µL frag5_for (10 mM)
  ·       10 µL 5x Phusion Buffer
  ·       27 µL nucleasefree H2O
  ·       2 µL dNTPs
  ·       2 µL DMSO
  ·       2 µL MgCl2 (50 mM)
  ·       4 µL Pfu-Polymerase
   
  ·       PCR program (30 cycles)
  • initial denaturation 98°C, 60s
  • denaturation 98°C, 30s
  • annealing 55°C, 30s
  • elongation 72°C, 300s
  • final elongation 72°C, 600s
   
  ·       preparative 1% agarose gel
  ·       gel displays the desired band of ~2500 bp as well as byproducts
   
26.08 Purification of TLO
  ·       using Wizard SV Gel and PCR Clean-Up System (Promega)
  ·       TLO c = 19,7 ng/µL
   
26.08 Amplification of TLO
  ·       PCR mixture (50 µL total volume)
  ·       1 µL TLO (c = 19,7 ng/µL)
  ·       1 µL frag5_rev (10 mM)
  ·       1 µL frag5_for (10 mM)
  ·       10 µL 5x Phusion Buffer
  ·       27 µL nucleasefree H2O
  ·       2 µL dNTPs
  ·       2 µL DMSO
  ·       2 µL MgCl2 (50 mM)
  ·       4 µL Pfu-Polymerase
   
  ·       PCR program (35 cycles)
  • initial denaturation 98°C, 60s
  • denaturation 98°C, 30s
  • annealing 55°C, 30s
  • elongation 72°C, 300s
  • final elongation 72°C, 600s
   
  ·       preparative 1% agarose gel
  ·       no distinctive bands
   
27.08 Amplification of TLO
  ·       PCR mixture (50 µL total volume)
  ·       1 µL TLO (c = 19,7 ng/µL, 1:4 dilution, final amount = 5 ng)
  ·       1 µL frag5_rev (10 mM)
  ·       1 µL frag5_for (1:10)
  ·       10 µL 5x Phusion Buffer
  ·       25,5 µL nucleasefree H2O
  ·       2 µL dNTPs (10 mM)
  ·       2,5 µL DMSO
  ·       2 µL MgCl2 (50 mM)
  ·       4 µL Pfu-Polymerase
  ·       2 µL BSA (10 mg/mL, final concentration = 0,4 µg/µL)
   
  ·       PCR program (35 cycles)
  ·       initial denaturation 98°C, 60s
  ·       denaturation 98°C, 30s
  ·       annealing 55°C, 30s
  ·       elongation 72°C, 480s
  ·       final elongation 72°C, 600s
   
  ·       preparative 1% agarose gel
  ·       no distinctive bands (just like on 26.08)
   
28.08 Amplification of TLO
  ·       PCR mixture (50 µL total volume)
  ·       1 µL TLO (c = 19,7 ng/µL, 1:20 dilution, final amount = 1 ng)
  ·       2,5 µL primer frag5_rev (10 mM)
  ·       2,5 µL primer frag5_for (10 mM)
  ·       19 µL nucleasefree H2O
  ·       25 µL 2x Q5 Mastermix (NEB)
   
  ·       PCR program (35 cycles)
  ·       initial denaturation 98°C, 60s
  ·       denaturation 98°C, 30s
  ·       annealing 66°C, 30s
  ·       elongation 72°C, 90s
  ·       final elongation 72°C, 120s
   
  ·       preparative 1% agarose gel
  ·     no distinctive bands (just like on 26.08) à probably the template is poorly
   
28.08 Amplification of TLO
  ·       PCR mixture (50 µL total volume)
  ·       1 µL TLO gBLOCK assembly (c = 34,4 ng/µL, 1:30 dilution, final amount = 1 ng)
  ·      
  2,5 µL primer frag5_rev (10 mM)
  ·       2,5 µL primer frag5_for (10 mM)
  ·       19 µL nucleasefree H2O
  ·       25 µL 2x Q5 Mastermix (NEB)
   
  ·       PCR program (35 cycles)
  ·       initial denaturation 98°C, 60s
  ·       denaturation 98°C, 30s
  ·       annealing 66°C, 30s
  ·       elongation 72°C, 90s
  ·       final elongation 72°C, 120s
   
  ·       preparative 1% agarose gel
  ·       gel displays the desired band of ~2500 bp as well as byproducts
   
29.08 Purification of TLO
  ·       using Wizard SV Gel and PCR Clean-Up System (Promega)
  ·       TLO c = 241,7 ng/µL 260/280 = 1,93 260/230 = 2,02
   
31.08 InFusion
  ·       reaction mixture (20 µL total volume)
  ·       1µl pBAD1 (130 bp, c = 45 ng/µL, 45 ng)
  ·       1µl pBAD4 (130 bp, c = 57,7 ng/µL, 58 ng)
  ·       1µl terminator (100 bp, c = 44 ng/µL, 44 ng)
  ·       3,5µl TLO (2440 bp, c = 241,7 ng/µL, 780 ng)
  ·       4,5µl CMK (1850 bp, c = 154,2 ng/µL, 680 ng)
  ·       3,5µl pSB1C3 (2100 bp, c = 120,8 ng/µL, 360 ng)
  ·       4µl 5x InFusion Pfu MasterMix
  ·       1,5µl nucleasefree H2O
   
  ·       treatment of the reaction mixture according to InFusion protocol
   
  ·       transformation into E.coli Top10 according to heat shock protocol
  ·       2 colonies grew on LB-Cam plates, most likely the reaction volume was to high
   
2.09 Control of pSB1C3-CMK-TLO clone 1 and 2 using colony PCR and test restriction
  ·       Colony PCR
   
  ·       PCR mixture (50 µL total volume)
  ·       1µl VR primer (10 mM)
  ·       1µl VF2 primer (10 mM)
  ·       5µl Colony LB Medium (Colony 1/Colony 2)
  ·       2µl MgCl2
  ·       1µl dNTP Mix
  ·       1µl DMSO
  ·       5µl 10x Taq Buffer
  ·       1µl Taq-Polymerase
  ·       33µl nucleasefree H2O
   
  ·       PCR Programm
  ·       initial denaturation 300s 95°C
  ·       denaturation 30s 95°C
  ·       annealing 30s 55°C
  ·       elongation 150s 72°C
  ·       final elongation 300s 72°C
   
   
  ·       Purification using Pure Yield Plasmid Miniprep System (Promega)
  ·         Colonie 1 = pSB1C3-TLO-CMK 1
  o    c = 245,5 ng/ul
  o    260/280 = 1,87
  o    260/230 = 2,23
  ·         Colonie 2 = pSB1C3-TLO-CMK 2
  o    c = 107,7 ng/ul
  o    260/280 = 1,95
  o    260/230 = 2,28
   
  ·       digestion with EcoRI and double digest with EcoRI and PstI
   
  ·       analytical 1% agarose gel
  ·       pattern is as expected
  ·       colony PCR product is off by 2000 bp
  ·       linearized plasmid is off by 1000 bp
   
   
3.09 Control of pSB1C3-CMK-TLO clone 1 and 2 using PCR
  ·        PCR mixture (50 µL total volume)
  ·         1µl forward primer
  ·         1µl reverse primer
  ·         1µl template
  ·         2µl MgCl2
  ·         1µl dNTP Mix
  ·         1µl DMSO
  ·         5µl 10x Taq-Buffer
  ·         1µl Taq-Polymerase
  ·         37µl H2O
   
  ·       template and primer specifications
  ·         template = pSB1C3-TLO-CMK 1, c = 245,5 ng/µl, 1:10 dilution
  o    PCR tube 1: frag1_for + frag4_rev
  o   
  PCR tube 2: frag2_for + frag2_rev
  o    PCR tube 3: frag3_for + frag3_rev
  o    PCR tube 4: frag4_for + frag4_rev
  o    PCR tube 5: frag5_for + frag5_rev
  ·         template = pSB1C3-TLO-CMK 2, c = 107,7 ng/µl, 1:4 dilution
  o    PCR tube 1: frag1_for + frag4_rev
  o    PCR tube 2: frag2_for + frag2_rev
  o    PCR tube 3: frag3_for + frag3_rev
  o    PCR tube 4: frag4_for + frag4_rev
  o    PCR tube 5: frag5_for + frag5_rev
   
  ·      PCR program (30 cycles)
  ·         initial denaturation 300s 95°C
  ·         denaturation 30s 95°C
  ·         annealing 30s 55°C
  ·         elongation 150s 72°C
  ·         final elongation 300s 72°C
   
  ·       analytical 1% agarose gel
  ·         besides many byproducts all fragments
  could be amplified from templates (framed bands)
  à clones are most likely correct
   
3.09 Induction of pSB1C3-TLO-CMK clones with L-Arabinose
  ·       induction with L-arabinose (0,02% w/v) at OD600 = 0,6
  ·       induced clones showed no difference to native E.coli Top10 cells when analyzed with an fluorospcetrometer
 
3.09 Sequencing of pSB1C3-TLO-CMK clones
  ·       were not able to sequence the whole insert, as pimers did not anneal sufficiently
  ·       results indicate that all five fragments were successfully ligated in the correct order
  ·       results show that gBLOCK assembly of TLO and CMK did not work correctly
   
3.09 Induction of pSB1C3-TLO-CMK clones with L-Arabinose
  ·       induction with L-arabinose (0,02% w/v) at OD600 = 0,6
  ·       induced clones showed no difference to native E.coli Top10 cells when analyzed with an fluorospcetrometer
 
3.09 Sequencing of pSB1C3-TLO-CMK clones
  ·       were not able to sequence the whole insert, as pimers did not anneal sufficiently
  ·       results indicate that all five fragments were successfully ligated in the correct order
  ·       results show that gBLOCK assembly of TLO and CMK did not work correctly