| 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
|
|
|
|
"
