Team:TU Darmstadt/labbook

From 2013.igem.org

(Difference between revisions)

Revision as of 09:48, 3 October 2013

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

@@ Line 98: / Line 98: @@
 !align="center"|[[Team:TU_Darmstadt/protocols|<span style="color:white;font-size:160%;"> Protocols |</span> ]]
 !align="center"|[[Team:TU_Darmstadt/materials|<span style="color:white;font-size:160%;"> Materials |</span>]]
+|}
+{| class="wikitable" <!-- generated with [[w:de:Wikipedia:Technik/Text/Basic/EXCEL-Tabellenumwandlung]] V1.9 -->
+|-   valign="top"
+| align="right" width="30" height="15" | 13.08
+|style="font-weight:bold" width="416" | gBLOCK assembly of CMK and TLO
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       PCR mixture
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       1 µL of each gBLOCK
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       CMK: B_01 – B_04
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       TLO: A_01 – A_06
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       10 µL 5x Q5 Reaction Buffer
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       2 µL dNTPs
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       1 µL Q5 Hot Start Polymerase
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       10 µL 5x Q5 High GC Enhancer
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       1 µL primer suffix-R (10 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       1 µL primer prefix_R (10 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| align="right" valign="top" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       PCR program (40 cycles)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       initial denaturation 94°C, 100s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       denaturation 94°C, 55s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       annealing 64°C, 55s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       elongation 72°C, 120s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       final elongation 72°C, 300s
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       preparative 1% agarose gel
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     gel displays bands of expected size à assembly was successful
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| align="right" valign="top" | &nbsp;
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+|style="font-weight:bold" align="right" | &nbsp;
+|-   valign="top"
+| align="right" height="15" | 13.08
+|style="font-weight:bold" | Purification
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       using Wizard SV Gel and PCR Clean-Up System (Promega)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       TLO c = 34,4 ng/µL
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       CMK c = 35,6 ng/µL
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-   valign="top"
+| align="right" height="15" | 19.08
+|style="font-weight:bold" | Overlap extension PCR
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | reaction mixture (50 µL total volume)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     25 µL Q5 High Fidelity 2x Master Mix (NEB)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     1 µL template
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     1 µL forward primer (10 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     1 µL reverse primer (10 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     22 µL nuclease-free H2O
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       template and primer specifications
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     pBAD1: pSB1C3[BBa_K206000] + frag1_for + frag4_rev
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     CMK: CMK gBLOCK assembly + frag2_for + frag2_rev
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     terminator: pSB1AK3[BBa_B0014] + frag3_for + frag3_rev
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     pBAD4: pSB1C3[BBa_K206000] + frag4_for + frag4_rev
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     TLO: TLO gBLOCK assembly + frag5_for + frag5_rev
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     pSB1C3: pSB1C3[fsC] + vector_for + vector_rev
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       PCR program (35 cycles)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     initial denaturation: 98°C, 60s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     denaturation: 98°C, 45s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     annealing: 60°C, 40s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     elongation: 72°C, 90s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     final elongation: 72°C, 300s
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       preparative 1% agarose gel
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     gel displays byproducts
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | à subsequent purification and amplification of desired bands (framed)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| align="right" valign="top" | &nbsp;
+|-   valign="top"
+| align="right" height="15" | 19.08
+|style="font-weight:bold" | Purification
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       using Wizard SV Gel and PCR Clean-Up System (Promega)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       pBAD1 c = 10 ng/µL
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       CMK c = 30 ng/µL
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       terminator c = 7 ng/µL
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       pBAD4 c = 11 ng/µL
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       TLO c = 22 ng/µL
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       pSB1C3 c = 16 ng/µL
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| align="right" valign="top" | &nbsp;
+|-   valign="top"
+| align="right" height="15" | 19.08
+|style="font-weight:bold" | Amplification PCR of pBAD1, terminator and pBAD4
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       reaction mixture (25 µL total volume)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     12,5 µL Q5 High Fidelity 2x Master Mix (NEB)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     1 µL template
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     1 µL forward primer (10 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     1 µL reverse primer (10 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     9,5 µL nuclease-free H2O
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       template and primer specifications
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     pBAD1: pBAD1 (c = 10 ng/µL, 19.08) + frag1_for + frag4_rev
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     terminator: terminator (c = 7 ng/µL, 19.08) + frag3_for + frag3_rev
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     pBAD4: pBAD4 (c = 11 ng/µL, 19.08)  + frag4_for + frag4_rev
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       PCR program (35 cycles)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·      initial denaturation: 98°C, 60s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·      denaturation: 98°C, 45s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·      annealing: 70°C, 30s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·      elongation: 72°C, 30s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·      final elongation: 72°C, 300s
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       analytical 1% agarose gel
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       pBAD1 shows single band of expected size, 70°C is the optimal annealing temperature
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       terminator and pBAD4 show no bands
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| align="right" valign="top" | &nbsp;
+|-   valign="top"
+| align="right" height="15" | 19.08
+|style="font-weight:bold" | Gradient PCR of the amplification of pBAD1, terminator and pBAD4
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       reaction mixture (25 µL total volume)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     12,5 µL Q5 High Fidelity 2x Master Mix (NEB)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     4 µL template
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     1 µL forward primer (10 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     1 µL reverse primer (10 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     6,5 µL nuclease-free H2O
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       template and primer specifications
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     pBAD1: pBAD1 (c = 10 ng/µL, 19.08) + frag1_for + frag4_rev
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     terminator: terminator (c = 7 ng/µL, 19.08) + frag3_for + frag3_rev
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     pBAD4: pBAD4 (c = 11 ng/µL, 19.08)  + frag4_for + frag4_rev
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       PCR program (35 cycles)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       initial denaturation 98°C, 60s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       denaturation 98°C, 45s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       annealing 55°C/57°C/61°C/65°C, 30s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       elongation 72°C, 30s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       final elongation 72°C, 300s
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       analytical 1% agarose gel
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       optimal annealing temperature for terminator is 55°C
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       optimal annealing temperature for pBAD4 is 55°C
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-   valign="top"
+| align="right" height="15" | 20.08
+|style="font-weight:bold" | Purification of PCR batches pBAD1 (Amplification PCR, 19.08), terminator 55°C (Gradient PCR, 19.08) and pBAD4 55°C (Gradient PCR, 19.08)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       using Wizard SV Gel and PCR Clean-Up System (Promega)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       pBAD1 c = 45 ng/µL
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       terminator c = 44 ng/µL 260/280 = 1,67 260/230 = 0,73
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       pBAD4 c = 57,7 ng/µL 260/280 = 1,81 260/230 = 0,67
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-   valign="top"
+| align="right" height="15" | 21.08
+|style="font-weight:bold" | Amplification PCR of CMK, TLO and pSB1C3
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       PCR mixture (50 µL total volume)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       25 µL Q5 High Fidelity 2x Master Mix
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       1 µL template
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       1 µL forward primer
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       1 µL reverse primer
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       22 µL nuclease-free H2O
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       template and primer specifications
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     CMK: CMK (c = 30 ng/µL, 19.08) + frag2_for + frag2_rev
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     TLO: TLO (c = 22 ng/µL, 19.08) + frag5_for + frag5_rev
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     pSB1C3: pSB1C3 (c = 16 ng/µL, 19.08) + vector_for + vector_rev
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       PCR program (35 cycles)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       initial denaturation 98°C, 60s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       denaturation 98°C, 45s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       annealing 55°C, 40s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       elongation 72°C, 90s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       final elongation 72°C, 300s
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       analytical 1% agarose gel
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       gel displays critical amount of byproducts
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-   valign="top"
+| align="right" height="15" | 21.08
+|style="font-weight:bold" | Gradient PCR of the amplification of CMK, TLO and pSB1C3
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       Amplification using Q5 Polymerase
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| align="right" valign="top" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       PCR mixture (50 µL total volume)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       25 µL Q5 High Fidelity 2xMaster Mix
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       2 µL template
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       1 µL forward primer (10 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       1 µL reverse primer (10 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       22 µL nuclease-free H2O
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       PCR program (35 cycles)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       initial denaturation 98°C, 60s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       denaturation 98°C, 45s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       annealing 55°C/56,7°C/60,4°C/62,9°C/64,9°C, 30s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       elongation 72°C, 30s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       final elongation 72°C, 300s
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       Amplification using Pfu Polymerase (homehold)
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       PCR mixture (50 µL total volume)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 2 µL template
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 10 µL 5x Phusion-Buffer
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 2 µL dNTPs
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 2 µL DMSO
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 4 µL Pfu-Polymerase
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 2 µL MgCl2 (50 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 1 µL forward primer (10 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 1 µL reverse primer (10 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 26 µL nuclease-free H2O
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       PCR program (30 cycles)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • initial denaturation 98°C, 60s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • denaturation 98°C, 30s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • annealing 55°C/56,4°C/59,3°C/62,1°C/65°C, 30s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • elongation 72°C, 720s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • final elongation 72°C, 600s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| align="right" valign="top" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       template and primer specifications
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     CMK: CMK (c = 30 ng/µL, 19.08) + frag2_for + frag2_rev
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     TLO: TLO (c = 22 ng/µL, 19.08) + frag5_for + frag5_rev
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     pSB1C3: pSB1C3 (c = 16 ng/µL, 19.08) + vector_for + vector_rev
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       preparative 1% agarose gel
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     amplification with Pfu Polymerase yields lesser byproducts than amplification with Q5 polymerase
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     for all three templates higher annealing temperatures diminish the amount of by products
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-   valign="top"
+| align="right" height="15" | 22.08
+|style="font-weight:bold" | Purification of CMK, TLO and pSB1C3 from preparative gel
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       using Wizard SV Gel and PCR Clean-Up System (Promega)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       CMK c = 154,2 ng/µL 260/280 = 1,92 260/230 = 1,88
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       TLO c = 37,9 ng/µL 260/280 = 1,90 260/230 = 0,60
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       pSB1C3 c = 120,8 ng/µL 260/280 = 1,89 260/230 = 1,64
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-   valign="top"
+| align="right" height="15" | 23.08
+|style="font-weight:bold" | Amplification of TLO
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       PCR mixture (50 µL total volume)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 2 µL TLO (c = 37,9 ng/µL, 22.08)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 10 µL 5x Phusion-Buffer
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 2 µL dNTPs
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 2 µL DMSO
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 4 µL Pfu-Polymerase
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 2 µL MgCl2 (50 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 1 µL frag5_for (10 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 1 µL frag5_rev (10 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 26 µL nuclease-free H2O
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| align="right" valign="top" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       PCR program (30 cycles)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • initial denaturation 98°C, 60s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • denaturation 98°C, 30s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • annealing 55°C, 30s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • elongation 72°C, 300s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • final elongation 72°C, 600s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| align="right" valign="top" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       preparative 1% agarose gel
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     gel displays no distinctive bands
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| align="right" valign="top" | &nbsp;
+|-   valign="top"
+| align="right" height="15" | 24.08
+|style="font-weight:bold" | Amplification of TLO
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       PCR mixture (50 µL total volume)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 1 µL TLO (c = 37,9 ng/µL, 22.08)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 10 µL 5x Phusion-Buffer
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 2 µL dNTPs
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 2 µL DMSO
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 4 µL Pfu-Polymerase
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 2 µL MgCl2 (50 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 1 µL frag5_for (10 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 1 µL frag5_rev (10 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 27 µL nuclease-free H2O
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       PCR program (30 cycles)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • initial denaturation 98°C, 60s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • denaturation 98°C, 30s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • annealing 56°C, 30s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • elongation 72°C, 720s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • final elongation 72°C, 600s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| align="right" valign="top" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       preparative 1% agarose gel
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     gel displays no distinctive bands (just like on 23.08) à probably the template is poorly
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| align="right" valign="top" | &nbsp;
+|-   valign="top"
+| align="right" height="15" | 24.08
+|style="font-weight:bold" | Amplification of TLO
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       PCR mixture (50 µL total volume)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 1 µL TLO gBLOCK assembly (c = 34,4 ng/µL)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 10 µL 5x Phusion-Buffer
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 2 µL dNTPs
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 2 µL DMSO
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 4 µL Pfu-Polymerase
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 2 µL MgCl2 (50 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 1 µL frag5_for (10 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 1 µL frag5_rev (10 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 27 µL nuclease-free H2O
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       PCR program (30 cycles)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • initial denaturation 98°C, 60s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • denaturation 98°C, 30s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • annealing 56°C, 30s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • elongation 72°C, 720s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • final elongation 72°C, 600s
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       preparative 1% agarose gel
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     gel displays no distinctive bands
+|-   valign="top"
+| align="right" height="15" | 25.08
+|style="font-weight:bold" | Amplification of TLO
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       PCR mixture (50 µL total volume)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 2 µL TLO gBLOCK assembly (c = 34,4 ng/µL)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 10 µL 5x Phusion-Buffer
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 2 µL dNTPs
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 2 µL DMSO
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 4 µL Pfu-Polymerase
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 2 µL MgCl2 (50 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 1 µL frag5_for (10 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 1 µL frag5_rev (10 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • 26 µL nuclease-free H2O
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       PCR program (30 cycles)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • initial denaturation 98°C, 60s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • denaturation 98°C, 30s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • annealing 55°C, 30s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • elongation 72°C, 300s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • final elongation 72°C, 600s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| align="right" valign="top" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       preparative 1% agarose gel
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       gel displays the desired band of ~2500 bp as well as byproducts
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-   valign="top"
+| align="right" height="15" | 25.08
+|style="font-weight:bold" | Purification of TLO
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       using Wizard SV Gel and PCR Clean-Up System (Promega)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       TLO c = 19 ng/µL
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+|style="font-weight:bold" align="right" | &nbsp;
+|-   valign="top"
+| align="right" height="15" | 25.08
+|style="font-weight:bold" | InFusion
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       reaction mixture (71 µL total volume)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       1µl pBAD1 (130 bp, c = 45 ng/µL, 45 ng)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       1µl pBAD4 (130 bp, c = 57,7 ng/µL, 58 ng)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       1µl terminator (100 bp, c = 44 ng/µL, 44 ng)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       42µl TLO (2440 bp, c = 19 ng/µL, 780 ng)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       4,5µl CMK (1850 bp, c = 154,2 ng/µL, 680 ng)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       3,5µl pSB1C3 (2100 bp, c = 120,8 ng/µL, 360 ng)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       18µl 5x InFusion Pfu MasterMix
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       treatment of the reaction mixture according to InFusion protocol
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       transformation into E.coli Top10 according to heat shock protocol
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       no colonies grew on LB-Cam plates, most likely the reaction volume was to high
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+|style="font-weight:bold" align="right" | &nbsp;
+|-   valign="top"
+| align="right" height="15" | 26.08
+|style="font-weight:bold" | Amplification of TLO
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·        PCR mixture (50 µL total volume)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | 1 µL TLO gBLOCK assembly (c = 34,4 ng/µL)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       1 µL frag5_rev (10 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       1 µL frag5_for (10 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       10 µL 5x Phusion Buffer
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       27 µL nucleasefree H2O
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       2 µL dNTPs
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       2 µL DMSO
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       2 µL MgCl2 (50 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       4 µL Pfu-Polymerase
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       PCR program (30 cycles)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • initial denaturation 98°C, 60s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • denaturation 98°C, 30s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • annealing 55°C, 30s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • elongation 72°C, 300s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • final elongation 72°C, 600s
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       preparative 1% agarose gel
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       gel displays the desired band of ~2500 bp as well as byproducts
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-   valign="top"
+| align="right" height="15" | 26.08
+|style="font-weight:bold" | Purification of TLO
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       using Wizard SV Gel and PCR Clean-Up System (Promega)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       TLO c = 19,7 ng/µL
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-   valign="top"
+| align="right" height="15" | 26.08
+|style="font-weight:bold" | Amplification of TLO
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       PCR mixture (50 µL total volume)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       1 µL TLO (c = 19,7 ng/µL)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       1 µL frag5_rev (10 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       1 µL frag5_for (10 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       10 µL 5x Phusion Buffer
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       27 µL nucleasefree H2O
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       2 µL dNTPs
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       2 µL DMSO
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       2 µL MgCl2 (50 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       4 µL Pfu-Polymerase
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| align="right" valign="top" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       PCR program (35 cycles)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • initial denaturation 98°C, 60s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • denaturation 98°C, 30s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • annealing 55°C, 30s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • elongation 72°C, 300s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | • final elongation 72°C, 600s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| align="right" valign="top" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       preparative 1% agarose gel
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       no distinctive bands
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-   valign="top"
+| align="right" height="15" | 27.08
+|style="font-weight:bold" | Amplification of TLO
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       PCR mixture (50 µL total volume)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       1 µL TLO (c = 19,7 ng/µL, 1:4 dilution, final amount = 5 ng)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       1 µL frag5_rev (10 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       1 µL frag5_for (1:10)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       10 µL 5x Phusion Buffer
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       25,5 µL nucleasefree H2O
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       2 µL dNTPs (10 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       2,5 µL DMSO
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       2 µL MgCl2 (50 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       4 µL Pfu-Polymerase
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       2 µL BSA (10 mg/mL, final concentration = 0,4 µg/µL)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| align="right" valign="top" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       PCR program (35 cycles)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       initial denaturation 98°C, 60s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       denaturation 98°C, 30s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       annealing 55°C, 30s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       elongation 72°C, 480s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       final elongation 72°C, 600s
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       preparative 1% agarose gel
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       no distinctive bands (just like on 26.08)
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-   valign="top"
+| align="right" height="15" | 28.08
+|style="font-weight:bold" | Amplification of TLO
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       PCR mixture (50 µL total volume)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       1 µL TLO (c = 19,7 ng/µL, 1:20 dilution, final amount = 1 ng)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       2,5 µL primer frag5_rev (10 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       2,5 µL primer frag5_for (10 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       19 µL nucleasefree H2O
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       25 µL 2x Q5 Mastermix (NEB)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| align="right" valign="top" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       PCR program (35 cycles)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       initial denaturation 98°C, 60s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       denaturation 98°C, 30s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       annealing 66°C, 30s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       elongation 72°C, 90s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       final elongation 72°C, 120s
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       preparative 1% agarose gel
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·     no distinctive bands (just like on 26.08) à probably the template is poorly
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+|style="font-weight:bold" align="right" | &nbsp;
+|-   valign="top"
+| align="right" height="15" | 28.08
+|style="font-weight:bold" | Amplification of TLO
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       PCR mixture (50 µL total volume)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       1 µL TLO gBLOCK assembly (c = 34,4 ng/µL, 1:30 dilution, final amount = 1 ng)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | 2,5 µL primer frag5_rev (10 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       2,5 µL primer frag5_for (10 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       19 µL nucleasefree H2O
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       25 µL 2x Q5 Mastermix (NEB)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| align="right" valign="top" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       PCR program (35 cycles)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       initial denaturation 98°C, 60s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       denaturation 98°C, 30s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       annealing 66°C, 30s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       elongation 72°C, 90s
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       final elongation 72°C, 120s
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       preparative 1% agarose gel
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       gel displays the desired band of ~2500 bp as well as byproducts
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+|style="font-weight:bold" align="right" | &nbsp;
+|-   valign="top"
+| align="right" height="15" | 29.08
+|style="font-weight:bold" | Purification of TLO
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       using Wizard SV Gel and PCR Clean-Up System (Promega)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       TLO c = 241,7 ng/µL 260/280 = 1,93 260/230 = 2,02
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+|style="font-weight:bold" align="right" | &nbsp;
+|-   valign="top"
+| align="right" height="15" | 31.08
+|style="font-weight:bold" | InFusion
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       reaction mixture (20 µL total volume)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       1µl pBAD1 (130 bp, c = 45 ng/µL, 45 ng)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       1µl pBAD4 (130 bp, c = 57,7 ng/µL, 58 ng)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       1µl terminator (100 bp, c = 44 ng/µL, 44 ng)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       3,5µl TLO (2440 bp, c = 241,7 ng/µL, 780 ng)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       4,5µl CMK (1850 bp, c = 154,2 ng/µL, 680 ng)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       3,5µl pSB1C3 (2100 bp, c = 120,8 ng/µL, 360 ng)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       4µl 5x InFusion Pfu MasterMix
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       1,5µl nucleasefree H2O
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       treatment of the reaction mixture according to InFusion protocol
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       transformation into E.coli Top10 according to heat shock protocol
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       2 colonies grew on LB-Cam plates, most likely the reaction volume was to high
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+|style="font-weight:bold" align="right" | &nbsp;
+|-   valign="top"
+| align="right" height="15" | 2.09
+|style="font-weight:bold" | Control of pSB1C3-CMK-TLO clone 1 and 2 using colony PCR and test restriction
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       Colony PCR
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| align="right" valign="top" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       PCR mixture (50 µL total volume)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       1µl VR primer (10 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       1µl VF2 primer (10 mM)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       5µl Colony LB Medium (Colony 1/Colony 2)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       2µl MgCl2
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       1µl dNTP Mix
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       1µl DMSO
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       5µl 10x Taq Buffer
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       1µl Taq-Polymerase
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       33µl nucleasefree H2O
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       PCR Programm
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       initial denaturation 300s 95°C
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       denaturation 30s 95°C
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       annealing 30s 55°C
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       elongation 150s 72°C
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       final elongation 300s 72°C
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       Purification using Pure Yield Plasmid Miniprep System (Promega)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·         Colonie 1 = pSB1C3-TLO-CMK 1
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | o    c = 245,5 ng/ul
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | o    260/280 = 1,87
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | o    260/230 = 2,23
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·         Colonie 2 = pSB1C3-TLO-CMK 2
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | o    c = 107,7 ng/ul
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | o    260/280 = 1,95
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | o    260/230 = 2,28
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       digestion with EcoRI and double digest with EcoRI and PstI
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       analytical 1% agarose gel
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       pattern is as expected
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       colony PCR product is off by 2000 bp
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       linearized plasmid is off by 1000 bp
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| align="right" valign="top" | &nbsp;
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-   valign="top"
+| align="right" height="15" | 3.09
+|style="font-weight:bold" | Control of pSB1C3-CMK-TLO clone 1 and 2 using PCR
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·        PCR mixture (50 µL total volume)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·         1µl forward primer
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·         1µl reverse primer
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·         1µl template
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·         2µl MgCl2
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·         1µl dNTP Mix
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·         1µl DMSO
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·         5µl 10x Taq-Buffer
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·         1µl Taq-Polymerase
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·         37µl H2O
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| align="right" valign="top" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       template and primer specifications
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·         template = pSB1C3-TLO-CMK 1, c = 245,5 ng/µl, 1:10 dilution
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | o    PCR tube 1: frag1_for + frag4_rev
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | o
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | PCR tube 2: frag2_for + frag2_rev
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | o    PCR tube 3: frag3_for + frag3_rev
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | o    PCR tube 4: frag4_for + frag4_rev
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | o    PCR tube 5: frag5_for + frag5_rev
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·         template = pSB1C3-TLO-CMK 2, c = 107,7 ng/µl, 1:4 dilution
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | o     PCR tube 1: frag1_for + frag4_rev
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | o    PCR tube 2: frag2_for + frag2_rev
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | o    PCR tube 3: frag3_for + frag3_rev
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | o    PCR tube 4: frag4_for + frag4_rev
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | o    PCR tube 5: frag5_for + frag5_rev
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·      PCR program (30 cycles)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·         initial denaturation 300s 95°C
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·         denaturation 30s 95°C
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·         annealing 30s 55°C
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·         elongation 150s 72°C
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·         final elongation 300s 72°C
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       analytical 1% agarose gel
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·         besides many byproducts all fragments
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | could be amplified from templates (framed bands)
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | à clones are most likely correct
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-   valign="top"
+| align="right" height="15" | 3.09
+|style="font-weight:bold" | Induction of pSB1C3-TLO-CMK clones with L-Arabinose
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       induction with L-arabinose (0,02% w/v) at OD600 = 0,6
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       induced clones showed no difference to native E.coli Top10 cells when analyzed with an fluorospcetrometer
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+ |
+|-   valign="top"
+| align="right" height="15" | 3.09
+|style="font-weight:bold" | Sequencing of pSB1C3-TLO-CMK clones
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       were not able to sequence the whole insert, as pimers did not anneal sufficiently
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       results indicate that all five fragments were successfully ligated in the correct order
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       results show that gBLOCK assembly of TLO and CMK did not work correctly
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
+|-   valign="top"
+| align="right" height="15" | 3.09
+|style="font-weight:bold" | Induction of pSB1C3-TLO-CMK clones with L-Arabinose
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       induction with L-arabinose (0,02% w/v) at OD600 = 0,6
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       induced clones showed no difference to native E.coli Top10 cells when analyzed with an fluorospcetrometer
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+ |
+|-   valign="top"
+| align="right" height="15" | 3.09
+|style="font-weight:bold" | Sequencing of pSB1C3-TLO-CMK clones
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       were not able to sequence the whole insert, as pimers did not anneal sufficiently
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       results indicate that all five fragments were successfully ligated in the correct order
+|-
+| align="right" height="15"  valign="top" | &nbsp;
+| valign="top" | ·       results show that gBLOCK assembly of TLO and CMK did not work correctly
+|-   valign="top"
+| align="right" height="15" | &nbsp;
+| align="right" | &nbsp;
 |}