| 13.08 |
gBLOCK assembly of CMK and TLO |
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- 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, therefore assembly was successful
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| 13.08 |
Purification |
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- using Wizard SV Gel and PCR Clean-Up System (Promega)
- TLO c = 34,4 ng/µL
- CMK c = 35,6 ng/µL
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| 19.08 |
Overlap extension PCR |
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- 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
- subsequent purification and amplification of desired bands (framed)
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| 19.08 |
Purification |
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- 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
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| 19.08 |
Amplification PCR of pBAD1, terminator and pBAD4 |
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- 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
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| 19.08 |
Gradient PCR of the amplification of pBAD1, terminator and pBAD4 |
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- 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
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| 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) |
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- 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
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| 21.08 |
Amplification PCR of CMK, TLO and pSB1C3 |
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- 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
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| 22.08 |
Purification of CMK, TLO and pSB1C3 from preparative gel |
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- 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
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| 23.08 |
Amplification of TLO |
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- 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
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| 24.08 |
Amplification of TLO |
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- 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)
- probably the template is poorly
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| 24.08 |
Amplification of TLO |
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- 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
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| 25.08 |
Amplification of TLO |
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- 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
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| 25.08 |
Purification of TLO |
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- using Wizard SV Gel and PCR Clean-Up System (Promega)
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| 25.08 |
InFusion |
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- 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
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| 26.08 |
Amplification of TLO |
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- 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
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| 26.08 |
Purification of TLO |
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- using Wizard SV Gel and PCR Clean-Up System (Promega)
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| 26.08 |
Amplification of TLO |
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- 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
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| 27.08 |
Amplification of TLO |
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- 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)
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| 28.08 |
Amplification of TLO |
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- 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)
- probably the template is poorly
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| 28.08 |
Amplification of TLO |
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- 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
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| 29.08 |
Purification of TLO |
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- using Wizard SV Gel and PCR Clean-Up System (Promega)
- TLO c = 241,7 ng/µL 260/280 = 1,93 260/230 = 2,02
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| 31.08 |
InFusion |
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- 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
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| 02.09 |
Control of pSB1C3-CMK-TLO clone 1 and 2 using colony PCR and test restriction |
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- 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
- c = 245,5 ng/ul
- 260/280 = 1,87
- 260/230 = 2,23
- Colonie 2 = pSB1C3-TLO-CMK 2
- c = 107,7 ng/ul
- 260/280 = 1,95
- 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
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| 03.09 |
Control of pSB1C3-CMK-TLO clone 1 and 2 using PCR |
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- 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
- PCR tube 1: frag1_for + frag4_rev
- PCR tube 2: frag2_for + frag2_rev
- PCR tube 3: frag3_for + frag3_rev
- PCR tube 4: frag4_for + frag4_rev
- PCR tube 5: frag5_for + frag5_rev
- template = pSB1C3-TLO-CMK 2, c = 107,7 ng/µl, 1:4 dilution
- PCR tube 1: frag1_for + frag4_rev
- PCR tube 2: frag2_for + frag2_rev
- PCR tube 3: frag3_for + frag3_rev
- PCR tube 4: frag4_for + frag4_rev
- 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)
- clones are most likely correct
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| 03.09 |
Induction of pSB1C3-TLO-CMK clones with L-Arabinose |
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- 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
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| 03.09 |
Sequencing of pSB1C3-TLO-CMK clones |
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- 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
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