Team:Bielefeld-CeBiTec/Notebook/Labjournal

Labjournal

2017-04-03 - 2017-04-09

getting positive clones for further work

Investigators: Yannic Kerkhoff

Aim of the experiment: electroporation transformation of BBa_K1416000

Procedure:

  • standard electroporation protocol
    • used electro competend DH5alpha from NEB
    • streaked out on LB-plates with Cam

getting positive clones for further work

Investigators: Yannic Kerkhoff

Aim of the experiment: electroporation transformation of BBa_KJ36848

Procedure:

  • standard electroporation protocol
    • used electro competend DH5alpha from NEB
    • streaked out on LB-plates with Cam

multiplication of plasmids containing the Biobrick

Investigators: Yannic Kerkhoff

Aim of the experiment: preculture of transformed BBa_K1416000

Procedure:

  • standard procedure
    • 1,25֬ Cam in 5mL LB

2017-04-10 - 2017-04-16

getting plasmids containing the Biobrick for further experiments

Investigators: Yannic Kerkhoff

Aim of the experiment: plasmid isolation of preculture of BBa_K1416000

Procedure:

  • standard protocol
    • 517,9 ng/֌

multiplication of plasmids containing the Biobrick

Investigators: Yannic Kerkhoff

Aim of the experiment: preculture of transformed BBa_J36848

Procedure:

  • standard procedure
    • 1,25֬ Cam in 5mL LB

getting plasmids containing the Biobrick for further experiments

Investigators: Yannic Kerkhoff

Aim of the experiment: plasmid isolation of preculture of BBa_J36848

Procedure:

  • standard protocol
    • 231,8 ng/֌

2017-05-29 - 2017-06-04

getting positive clones for further work

Investigators: Yannic Kerkhoff

Aim of the experiment: heatshock transformation of BBa_E0400

Procedure:

  • standard heatshock protocol
    • used own produced chemo competend DH5alpha
    • streaked out on LB-plates with Amp

2017-06-05 - 2017-06-11

multiplication of plasmids containing the Biobrick

Investigators: Yannic Kerkhoff

Aim of the experiment: preculture of transformed BBa_E0400

Procedure:

  • standard procedure
    • 5,0֬ Amp in 5mL LB

getting plasmids containing the Biobrick for further experiments

Investigators: Yannic Kerkhoff

Aim of the experiment: plasmid isolation of preculture of BBa_E0400

Procedure:

  • standard protocol
    • 88,4 ng/֌

getting positive clones for further work

Investigators: Yannic Kerkhoff

Aim of the experiment: heatshock transformation of BBa_K525998

Procedure:

  • standard heatshock protocol
    • used own produced chemo competend DH5alpha
    • streaked out on LB-plates with Cam

multiplication of plasmids containing the Biobrick

Investigators: Yannic Kerkhoff

Aim of the experiment: preculture of transformed BBa_K525998

Procedure:

  • standard procedure
    • 1,25֬ Cam in 5mL LB

getting plasmids containing the Biobrick for further experiments

Investigators: Yannic Kerkhoff

Aim of the experiment: plasmid isolation of preculture of BBa_K525998

Procedure:

  • standard protocol
    • 140,9 ng/֌

2017-06-12 - 2017-06-18

Assemble the fragments to get the composite Part BBa_P1:GLS1

Investigators: Yannic Kerkhoff

Aim of the experiment: Gibson assembly of F1,F3,F5

Procedure:

To get and purified the plasmid

Investigators: Christina Drake

Aim of the experiment: Plasmidisolation of barnase-plasmid

Procedure:

  • protocol from Macherley-Nagel: Plasmid DNA purification, protocol 5: NucleoSpin
  • concentration measurement by nanoprop
  • sequenzingorder

Duplicate the parts

Investigators: Christina Drake, Denise Kerkhoff

Aim of the experiment: Transformation via Distribution of the parts BBA_J04450, BBa_I746909, BBa_K80800 and BBa_psB1K5

Procedure:

Duplicate the part

Investigators: Christina Drake, Denise Kerkhoff

Aim of the experiment: Transformation of BBa_psB6A1

Procedure:

Cellgrowth with the plasmids

Investigators: Denise Kerkhoff, Christina Drake

Aim of the experiment: Overnightculture of BBA_J04450, BBa_I746909 and BBa_K808000

Procedure:

  • 3 ml LB-medium and at BBA_J04450 3µl tetracycline and atBBa_I746909 and BBa_K808000 0,75 µl chloramphenicol
  • inkubation overnight 37°C, 140rpm

To get and purified the plasmid

Investigators: Christina Drake

Aim of the experiment: Plasmidisolation of BBA_J04450, BBa_I746909 and BBa_K80800-plasmids

Procedure:

  • protocol from Macherley-Nagel: Plasmid DNA purification, protocol 5: NucleoSpin
  • concentration measurement by nanoprop:
    • BBA_J04450 26ng/µl and 25,7ng/µl
    • BBa_I746909 90,2ng/µl
    • 104,3 ng/µl -BBa_K808000 118,3 ng/µl
  • and 146,6 ng/µl
  • sequenzingorder

2017-06-19 - 2017-06-25

Cellgrowth with the plasmid

Investigators: Denise Kerkhoff

Aim of the experiment: Overnightculture of BBA_psB3K5

Procedure:

  • 3 ml LB-medium and 2,5 µl kanamycin
  • inkubation overnight 37°C, 140rpm

getting positive clones for further work

Investigators: Yannic Kerkhoff

Aim of the experiment: heatshock transformation of assembled fragments F5,F2,F4

Procedure:

  • standard heatshock protocol
    • used own produced chemo competend DH5alpha
    • streaked out on LB-plates with Cam

multiplication, verification and purification of the needed fragment F2: GFPLinker2

Investigators: Yannic Kerkhoff

Aim of the experiment: Q5-PCR, gel and PCR-cleanUp of F2

Procedure:

  • standard Q5-PCR protocol
    • template: BBa_E0400
    • primer fwd: 17gk
    • primer rev: 17gn
    • annealing temperature: 58у
    • extension time: 50 seconds
  • standard PCR-cleanUp protocol
    • 125,8 ng/֌

multiplication, verification and purification of the needed fragment F4: StrepLinker2

Investigators: Yannic Kerkhoff

Aim of the experiment: Q5-PCR, gel and PCR-cleanUp of F4

Procedure:

  • standard Q5-PCR protocol
    • template: BBa_KJ36848
    • primer fwd: 17gp
    • primer rev: 17gq
    • annealing temperature: 61у
    • extension time: 30 seconds
  • standard PCR-cleanUp protocol
    • 127,0 ng/֌

multiplication, verification and purification of the needed fragment F5: linearized pSB1C3

Investigators: Yannic Kerkhoff

Aim of the experiment: Q5-PCR, gel and PCR-cleanUp of F5

Procedure:

  • standard Q5-PCR protocol
    • template: K1416000
    • primer fwd: 17gj
    • primer rev: 17gl
    • annealing temperature: 59у
    • extension time: 120 seconds
  • standard PCR-cleanUp protocol
    • 258,4 ng/֌

Assemble the fragments to get the composite Part BBa_P2:GLS2

Investigators: Yannic Kerkhoff

Aim of the experiment: Gibson assembly of F2,F4,F5

Procedure:

Screening for positive transformations containing the Biobrick BBa_P2:GLS2

Investigators: Yannic Kerkhoff

Aim of the experiment: GoTaq PCR of seven clones

Procedure:

  • standard GoTaq-protocol
    • template: colonie pcr clones of GA_P2:GLS2
    • primer fwd: Pr姩x_fwd
    • primer rev: Suffix_rev
    • annealing temperature: 56у
    • extension time: 40 seconds
  • positive clone 2

multiplication of plasmids containing the Biobrick

Investigators: Yannic Kerkhoff

Aim of the experiment: preculture of the positive clone 2 of BBa_P2:GLS2

Procedure:

  • standard procedure
    • 1,25֬ Cam in 5mL LB

getting plasmids containing the Biobrick for further experiments

Investigators: Yannic Kerkhoff

Aim of the experiment: plasmid isolation of preculture of BBa_P2:GLS2

Procedure:

  • standard protocol
    • 208,8 ng/֌

2017-06-26 - 2017-07-02

Check the sequence

Investigators: Christina Drake

Aim of the experiment: Sequenzingorder barnase

Procedure:

  • concentration measurement by nanoprop: 177 ng/µl
  • result: sequence was not barnase

PCR clean up of gblock1, gblock2 and pK18mobsacB-backbone

Investigators: Camilla Maerz

Aim of the experiment: Construction of pK18mobsacB_codA_del

Procedure:

Plasmidisolation and gibson assembly of pSB1C3-PlacUV5_PtNTT2 c30

Investigators: Camilla Maerz

Aim of the experiment: Construction of pSB1C3-PlacUV5_PtNTT2

Procedure:

Isolate T7RNAP from the chromosome of KRX-e.coli

Investigators: Christina Drake

Aim of the experiment: PCR of T7RNAP from KRX-e.coli

Procedure:

  • Q5 High-Fidelity PCR
    • annealingtemparatur 64°C, elogationtime 1 min
  • 1%-agarose-TAE-gel
    • 1kb Marker Geneuler and 5 µl sample with 1 µl 6*Loading Dye

Plated out Barnase from strain collection

Investigators: Christina Drake

Aim of the experiment: Plated out Barnase from strain collection

Procedure:

  • solved DNA in 100 µl SOC-medium
  • plated out on chloramphenicolplate
  • overnightincubation at 37°C

Colony PCR of pK18mobsacB-del_codA

Investigators: Camilla Maerz

Aim of the experiment: Construction of pK18mobsacB_codA_del

Procedure:

  • Go Tag (Promega) protocol
    • Primer: VR, VF2
    • Primer annealing: 56 у
    • Elongation: 140 s

2017-07-03 - 2017-07-09

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Investigators: Christina Drake

Aim of the experiment: Plasmidisolation of Barnase and colonies 1-5 T7RNAP

Procedure:

  • To get and purified the plasmid
    • protocol from Macherley-Nagel: Plasmid DNA purification, protocol 5: NucleoSpin
    • concentration measurement by nanoprop
  • >03.07.2017
  • tRNA
  • PCR of barnase
  • CDR
  • Check the correct barnasegen
    • Go-Taq-protocol
      • annealingtemparatur 58°C, elogationtime 30 s
    • 1%-agarose-TAE-gel
      • 100bp Marker Geneuler and 5 µl sample with 1 µl 6*Loading Dye
      • result: PCR-product could not be barnase

    2017-07-10 - 2017-07-16

    Biobrick Assembly of 2B1

    Investigators: Maximilian Edich

    Aim of the experiment: Biobrick Construction with RuBisCo Parts

    Procedure:

    • BioBrick Assembly
      • RuBisCoTAG mRFP as upstream part, T7-Terminator as downstream part, pSB1A3 as destination plasmid
    • transformation via heat shock and DH5α Cells
      • used 5µl from the ligation
    • plated out on Amp plates and let them grow over night

    Check the sequence

    Investigators: Christina Drake

    Aim of the experiment: Sequenzingorder barnase

    Procedure:

    • result: sequence was not barnase
  • >16.07.2017
  • tRNA
  • Overnightculture of TyrRS-Heidelberg-plasmid
  • CDR
  • Cellgrowth with the TyrRS-Heidelberg-plasmid
    • 3 ml LB-medium and 0,75 µl chloramphenicol
    • inkubation overnight 37°C, 140rpm

    Transformation of T7-Promotor

    Investigators: Maximilian Edich

    Aim of the experiment: Biobrick Construction with RuBisCo Parts

    Procedure:

    Preculture of colonies with T7-Promotor

    Investigators: Maximilian Edich

    Aim of the experiment: Biobrick Construction with RuBisCo Parts

    Procedure:

    2017-07-17 - 2017-07-23

    Isolate the tRNA

    Investigators: Christina Drake

    Aim of the experiment: PCR of TyrRS-Heidelberg-plasmid

    Procedure:

    Isolation of T7-Promotor

    Investigators: Laura Schlueter

    Aim of the experiment: Biobrick Construction with RuBisCo Parts

    Procedure:

    • Nucleospin Plasmidisolation protocol
      • got four products with the concentrations 50.1 ng/µl, 16.3 ng/µl, 24,2 ng/µl and 2.1 ng/µl

    Colony PCR with 2B1 Colonies

    Investigators: Maximilian Edich

    Aim of the experiment: Biobrick Construction with RuBisCo Parts

    Procedure:

    Biobrickassembly of 2B2 to 2B8, B1 and A1

    Investigators: Maximilian Edich

    Aim of the experiment: Biobrick Construction with RuBisCo Parts

    Procedure:

    • BioBrick Assembly
      • digest and ligate RuBisCo parts as upstreampart and T7-Terminator/Promotor as downstreampart and pSB1A3 as destination plasmid
        • ligate TAG2 and T7-Terminator to 2B2
        • ligate TAG111 and T7-Terminator to 2B3
        • ligate TAG474 and T7-Terminator to 2B4
        • ligate TAG2+TAG111 and T7-Terminator to 2B5
        • ligate TAG2+TAG474 and T7-Terminator to 2B6
        • ligate TAG111+TAG474 and T7-Terminator to 2B7
        • ligate TAG2+TAG111+TAG474 and T7-Terminator to 2B8
        • ligate RuBisCo mRFP and T7-Terminator to B1
        • ligate Carboxysome and T7-Promotor to A1
      • preculture of positive 2B1 colony

    Transformation of test devices

    Investigators: Camilla Maerz

    Aim of the experiment: Interlab study

    Procedure:

    • dilute test devives (distribution plate 6 & 7) in 10 ֌ ddH20
    • Transformation

    Transformation of 2B2-2B8, B1 and A1

    Investigators: Maximilian Edich

    Aim of the experiment: Biobrick Construction with RuBisCo Parts

    Procedure:

    Plasmidisolation of 2B1

    Investigators: Maximilian Edich

    Aim of the experiment: Biobrick Construction with RuBisCo Parts

    Procedure:

    • Nucleospin Plasmidisolation protocol
      • got 47 ng/µl and 41 ng/µl

    Reverse the insert

    Investigators: Christina Drake

    Aim of the experiment: PCR of T7GFP

    Procedure:

    • Q5 High-Fidelity PCR
      • Insert: annealingtemparatur 60°C,
      • Backbone:elogationtime 30s: annealingtemparatur 60°C, elogationtime 1 min
      • 1%-agarose-TAE-gel
    • result: PCR didn`t work

    Reverse the insert

    Investigators: Christina Drake

    Aim of the experiment: PCR of T7GFP

    Procedure:

    Preparation of over-night culture

    Investigators: Camilla Maerz

    Aim of the experiment: Interlab study

    Procedure:

    • Over-night culture of negative control, positive control, test device 1-6
      • LB-media + Cm were inoculated
      • Cultures were incubated over night (37у, 200 rpm)

    2017-07-24 - 2017-07-30

    Repeat the degestion and transformation from the 18th and 19th july

    Investigators: Maximilian Edich

    Aim of the experiment: Biobrick Construction with RuBisCo Parts

    Procedure:

    Cellgrowth with the plasmid

    Investigators: Christina Drake

    Aim of the experiment: Overnightculture of tRNA-psB1C3

    Procedure:

    • 3 ml LB-medium and 0,75 µl chloramphenicol
    • inkubation overnight 37°C, 140rpm
  • >31.07.2017
  • tRNA
  • Aquacloning of tRNA and backbone
  • CDR
  • construct a biobrick
    • transformation via heatschock

    Cloning of the ori (pSB6A1) in pSB1C3

    Investigators: Laura Schlueter

    Aim of the experiment: aaRS Plasmid (without aaRS)

    Procedure:

    • Gibson Assembly Master Mix
      • Backbone: 3,28 ֌ Ori 1.0: 1,73 ֌
      • Backbone: 0,32 ֌ Ori 1.1: 4,68 ֌
      • Backbone: 0,90 ֌ Ori 3.0: 4,10 ֌
    • Trafo: Hitzeschock
    • not successfull because of the wrong overlapps (try again)

    Colony PCR of the Retrafo of pRS (because of mixed culture)

    Investigators: Laura Schlueter

    Aim of the experiment: aaRS plasmid

    Procedure:

    • Sequencing was not succesfull (maybe mixed colony), so I did a retrafo with the aim of a successful sequencing (incorporation of the aaRS in pSB1C3)
      • pRS 3: 1-5
      • pRS 4: 1-5
      • pRS 5: 1-5
      • pRS 7: 1-5
      • no positive result (no band at 1000 bp)

    Amplification of pSB1C3 for the cloning of the ori in pSB1C3

    Investigators: Laura Schlueter

    Aim of the experiment: aaRS plasmid

    Procedure:

    • PCR with Q5 High-Fidelity Polymerase Master Mix, Primer jb,jn, 65у
    • gelelectrophoresis, 1% Agarose
    • band at 2000-2500 bp (supposed to be around 2200 bp)
    • Clean up from the gel
      • pSB1C3(nur ori): 22,2 ng/֌

    Amplification of pSB1C3 fragments for a 4 part Gibson (see page 4 in the paper-labbook)

    Investigators: Laura Schlueter

    Aim of the experiment: aaRS plasmid

    Procedure:

    • PCR, Q5 Master Mix
      • Primer jj, jn for fragment 2, 65у
      • Primer jb, ht for fragment 4, 65у
    • gelelectrophoresis, 1% Agarose
      • band at 300 (fragment 2, supposed to be around 326 bp)
      • band at 1200 (fragment 4, supposed to be around 1233 bp)
    • Clean up from the gel
      • fragment 2: 18,5 ng/֌ (overlapp(aaRS)_pSB1C3_overlapp(ori))
      • fragment 4: 19,1 ng/֌ (overlapp(ori)_pSB1C3_overlapp(aaRS))

    Colony PCR of 2B2-2B8, B1 and A1

    Investigators: Maximilian Edich

    Aim of the experiment: Biobrick Construction with RuBisCo Parts

    Procedure:

    • GoTaq® G2 PCR
    • most have negative results -> create new destination plasmid pSB1A3

    Colony PCR of retrafo and more colonies of the pRS

    Investigators: Laura Schlueter

    Aim of the experiment: aaRS plasmid

    Procedure:

    • GoTaq Master Mix, Primer vr,vf
      • 2 strong bands at 400 bp and 600 bp, small bands of pRS3,6 and pRS4,9 at around 1200-1500 bp
      • bands of pRS31, pRS32, pRS34, pRS36, pRS39 at around 1200-1500 bp
    • GoTaq Master Mix, Primer hq, jk (Gibson Assembly primer) with the colonies pRS31, pRS32, pRS34, pRS36, pRS39
      • pRS31, pRS32, pRS39 : stron bands at around 1200 bp
    • because of the the two positive colony PCRs (vf-vr, hq-jk) for these probes, the successful integration of the Tyr-aaRS in pSB1C3 seems to be successfull
    • pRS31, pRS32, pRS39 send to sequencing

    pRS31, pRS32, pRS39 plasmid isolation

    Investigators: Laura Schlueter

    Aim of the experiment: aaRS plasmid

    Procedure:

    • Macherey-Nagel purification kit
      • pRS31: 324,1 ng/֌
      • pRS32: 231,8 ng/֌
      • pRS39: 225,5 ng/֌
    • send to the sequencing
    • pRS31, pRS39 have the pSB1C3 with the Tyr-aaRS incorporated (but on position 32, the Arginine (cgt) should be changed to an Leucine (ctg), but it is not. On position 290 there really is a missing Leu, on position 268 the Asparagin is changed to an Arginine)

    2017-07-31 - 2017-08-06

    plasmid isolation of the positive colonies of the retrafo pRS4,4 (wrong!) and pRS3,12

    Investigators: Laura Schlueter

    Aim of the experiment: aaRS plasmid

    Procedure:

    • Macherey-Nagel purification kit with each 3 mL culture
      • pRS4,4: 44 ng/֌
      • pRS3,12: 68,3 ng/֌
      • not send to sequencing jet

    4 part Gibson and Trafo

    Investigators: Laura Schlueter

    Aim of the experiment: aaRS plasmid

    Procedure:

    • Gibson Assembly Master Mix with 5 ֬ template
      • pSB1C3 fragment (2): 18,5 ng/֌ around 350 bp 2,2 ֌
      • pSB1C3 fragment (4): 19,1 ng/֌ around 1300 bp 0,8 ֌
      • oK1.1 (pMB1 from pSB6A1): 5,8 ng/֌ around 1300 bp 1,8 ֌
      • Tyr-aaRS: 68,5 ng/֌ around 1000 bp 0,2 ֌
    • Trafo via heatshock

    Gibson of the ori in pSB1C3 (pori) and Trafo

    Investigators: Laura Schlueter

    Aim of the experiment: aaRS plasmid

    Procedure:

    • Gibson Assembly Master Mix with 5 ֌ template
      • pSB1C3(nur ori): 22,2 ng/֌ around 2100 bp 1,57 ֌
      • ok1.0: 5,80 ng/֌ around 1258 bp 3,43 ֌
    • Trafo via heatshock

    Transfer positive colonies

    Investigators: Maximilian Edich

    Aim of the experiment: Biobrick Construction with RuBisCo Parts

    Procedure:

    • picked and transfered positiv colonies of 2B2, 2B6, 2B7, 2B8 and A1 onto a new plate
    • incubate over night
    • 2B2 and 2B8 are contaminated with negativ colonies

    construct a biobrick

    Investigators: Christina Drake

    Aim of the experiment: Aquacloning of tRNA and backbone

    Procedure:

    • transformation via heatschock

    construct a biobrick

    Investigators: Christina Drake

    Aim of the experiment: Gibbsonassembly of barnase and backbone

    Procedure:

    • transformation via heatschock
  • >03.08.2017
  • tRNA
  • Overnightculture of tRNA-psB1C3 and barnase-psB1C3
  • CDR
  • Cellgrowth with the plasmid
    • 3 ml LB-medium and 0,75 µl chloramphenicol
    • inkubation overnight 37°C, 140rpm

    Preculture of 2B6, 2B7 and A1

    Investigators: Maximilian Edich

    Aim of the experiment: Biobrick Construction with RuBisCo Parts

    Procedure:

    Plasmidisolation

    Investigators: Maximilian Edich

    Aim of the experiment: Biobrick Construction with RuBisCo Parts

    Procedure:

    • Nucleospin Plasmidisolation protocol
      • got 67.3 ng/µl of 2B6
      • got 32.7 ng/µl of 2B7
      • got 51.6 ng/µl of A1

    To get and purified the plasmid

    Investigators: Christina Drake

    Aim of the experiment: Plasmidisolation of barnase-plasmid and tRNA-plasmids

    Procedure:

    • protocol from Macherley-Nagel: Plasmid DNA purification, protocol 5: NucleoSpin
    • concentration measurement by nanoprop
    • sequenzingorder

    Redo digestion of the destination plasmid

    Investigators: Maximilian Edich

    Aim of the experiment: Biobrick Construction with RuBisCo Parts

    Procedure:

    • analyze the destination plasmid digestion via gelelectrophoresis
      • pSB1A3 digestion was not effective
    • redo the digestion and analyze it on the gel
      • new dgestion was very effective

    2017-08-07 - 2017-08-13

    Amplification of the backbone (pSB1C3) and insert (Tyr-aaRS)

    Investigators: Laura Schlueter

    Aim of the experiment: aaRS plasmid

    Procedure:

    • PCR Q5 Polymerase (no Master Mix)
      • for 50 ֌ reaction
        • 10 ֌ Reaction Buffer
        • 5 ֌ 2mM dNTPs
        • 1 ֌ Template
        • 0,5 ֌ High Fidelity DNA Polymerase
        • 10,0 ֌ High GC Enhancer
        • 18,5 ֌ ddest H2O
        • 5,0 ֌ Primer
          • pSB1C3 (from RuBisCo, ncAA team): ht, jk 65у
          • aaRS (from Heidelberg) : hq, jk 65у (mistake! should be 58у, redone identically with 58у)
        • digestion of the PCR products with Dpn1: 5 ֌ CutSmart Reaktionsbuffer and 1 ֌ Dpn1 per 50 ֌ PCR product, on 37у over night)
      • agarose-gelelectrophhoresis, 1% (bands of the right size)
    • No further use, because results of the sequencing which show that the integration of the incorporation of the Tyr-aaRS in pSB1C3 was already successful (30.06.2017)

    Ligation of 2B2-2B5 and B1

    Investigators: Maximilian Edich

    Aim of the experiment: Biobrick Construction with RuBisCo Parts

    Procedure:

    • Ligation protocol from the BioBrick Assembly
      • Variation: incubated 1h at RT instead of 10min

    characterisation of T7GFP revers

    Investigators: Christina Drake

    Aim of the experiment: preparation of KRX-competent cells

    Procedure:

    • heatschocktransformation of T7GFP and T7GFP revers in KRX
  • >08.08.2017
  • tRNA
  • PCR of T7RNA-Polymerase
  • CDR
  • construct a biobrick for the selectionplasmid

    • Amplification of the backbone (pRS31) and insert (ori from pSB6A1, oK1.0) for Gibson assembly

    Investigators: Laura Schlueter

    Aim of the experiment: aaRS plasmid

    Procedure:

    • PCR Q5 Polymerase (protocoll of 07.08.2017)
      • pRS31: primer jb, jn 65у
      • oK1: primer jl, jc 65у
    • digestion with Dpn1 (protocoll of 07.08.2017)
    • agarose-gelelectrophhoresis, 1%
      • band around 1200 bp (oK1.1) / 2500 bp (pRS31), positive

    Gibson Assembly and Trafo of the pRS31 and oK1.0

    Investigators: Laura Schlueter

    Aim of the experiment: aaRS plasmid

    Procedure:

    • agarose-gelelectrophhoresis, 1%
    • purification from the gel with the Macherey-Nagel purification kit
      • oK1.1: 22,5 ng/֌
      • pRS32: 9,5 ng/֌
    • Gibson Assembly Master Mix with:
      • 4,1 ֌ of oK1.1: around 2480 bp 9,5 ng/֌
      • 0,9 ֌ of pRS32: around 1250 bp 22,5 ng/֌
    • Trafo via heatshock

    getting positive clones for further work

    Investigators: Yannic Kerkhoff

    Aim of the experiment: heatshock transformation of assembled fragments F13 and NPA-RS gene synthesis

    Procedure:

    • standard heatshock protocol
      • used own produced chemo competend DH5alpha
      • streaked out on LB-plates with Cam

    multiplication, verification and purification of the needed fragment F13: linearized ONBY-Part with removal of the ONBY-RS

    Investigators: Yannic Kerkhoff

    Aim of the experiment: Q5-PCR, gel and PCR-cleanUp of F13

    Procedure:

    • standard Q5-PCR protocol
      • template: K1416000
      • primer fwd: 17hl
      • primer rev: 17mv
      • annealing temperature: 63у
      • extension time: 90 seconds
    • standard PCR-cleanUp protocol
      • 80,6 ng/֌

    Assemble the fragments to get the composite Part BBa_P7:NPA-RS

    Investigators: Yannic Kerkhoff

    Aim of the experiment: Gibson assembly of F13 and NPA-RS gene synthesis

    Procedure:

    getting positive clones for further work

    Investigators: Yannic Kerkhoff

    Aim of the experiment: heatshock transformation of assembled fragments F5,F1,F3

    Procedure:

    • standard heatshock protocol
      • used own produced chemo competend DH5alpha
      • streaked out on LB-plates with Cam

    multiplication, verification and purification of the needed fragment F1: GFPLinker1

    Investigators: Yannic Kerkhoff

    Aim of the experiment: Q5-PCR, gel and PCR-cleanUp of F1

    Procedure:

    • standard Q5-PCR protocol
      • template: BBa_E0400
      • primer fwd: 17gk
      • primer rev: 17gm
      • annealing temperature: 58у
      • extension time: 50 seconds
    • standard PCR-cleanUp protocol
      • 15,9 ng/֌

    multiplication, verification and purification of the needed fragment F3: StrepLinker1

    Investigators: Yannic Kerkhoff

    Aim of the experiment: Q5-PCR, gel and PCR-cleanUp of F3

    Procedure:

    • standard Q5-PCR protocol
      • template: BBa_KJ36848
      • primer fwd: 17go
      • primer rev: 17gq
      • annealing temperature: 61у
      • extension time: 30 seconds
    • standard PCR-cleanUp protocol
      • 17,9 ng/֌

    Biobrickassembly

    Investigators: Maximilian Edich

    Aim of the experiment: Biobrick Construction with RuBisCo Parts

    Procedure:

    • BioBrick Assembly
    • repeat digestion of T7-Terminator downstrampart
    • ligate over night

    Transformation of new plasmids

    Investigators: Maximilian Edich

    Aim of the experiment: Biobrick Construction with RuBisCo Parts

    Procedure:

    Primer annealing preparation

    Investigators: Camilla Maerz

    Aim of the experiment: M.A.X restriction system - Primer annealing test

    Procedure:

    • Both Primers (17jh; 17ji) were resuspended in 1x Composite Buffer
    • Dilutions: 100 ֍, 10 ֍, 5 ֍, 2.5 ֍, 0.5 ֍
    • OD 260 were measured to determine the real concentration

    2017-08-14 - 2017-08-20

    Screening for positive transformations containing the Biobrick BBa_P2:GLS2

    Investigators: Yannic Kerkhoff

    Aim of the experiment: GoTaq PCR of 4 clones

    Procedure:

    • standard GoTaq-protocol
      • template: colonie pcr clones of GA_P1:GLS1
      • primer fwd: Pr姩x_fwd
      • primer rev: Suffix_rev
      • annealing temperature: 56у
      • extension time: 40 seconds
    • positive clone 3

    Primer annealing of 17jh & 17ji

    Investigators: Camilla Maerz

    Aim of the experiment: M.A.X restriction system - Primer annealing test

    Procedure:

    • Protocol for annealing oligonucleotides (sigmaaldrich)
      • Eqimolar Primers were mixed (50 ֌)
      • Thermal profile (thermocycler)
        • 95 у, 2 min
        • Cool to 25 у over 45 min
        • Cool to 4 у for temporary storage

    Primer annealing of 17jl & 17jm

    Investigators: Camilla Maerz

    Aim of the experiment: M.A.X restriction system - Primer annealing test

    Procedure:

    • Both Primers were resuspended in ddH20 to a final concentration of 100 ֍
    • HEPES annealing standard protocol
    • Aqua annealing standard protocol
      • Final volume: 20 ֌
    • Agarose gel electrophoresis (2%)
      • 60 V, 50 min

    Native PAGE of annealed primers (17jl & 17jm)

    Investigators: Camilla Maerz

    Aim of the experiment: M.A.X restriction system - Primer annealing test

    Procedure:

    • Native DNA PAGE standard protocol
      • tested samples:
        • HEPES annealing (17jl & 17jm): 100 ֍, 10 ֍, 5 ֍, 2.5 ֍, 1 ֍, 0.5 ֍
        • Aqua annealing (17jl & 17jm): 100 ֍, 10 ֍, 5 ֍, 2.5 ֍, 1 ֍, 0.5 ֍
      • Variation: ~40 mA

    2017-08-21 - 2017-08-27

    Native PAGE of annealed primers (17jl & 17jm)

    Investigators: Camilla Maerz

    Aim of the experiment: M.A.X restriction system - Primer annealing test

    Procedure:

    • Native DNA PAGE standard protocol
      • tested samples:
        • HEPES annealing (17jl & 17jm): 100 ֍, 10 ֍, 5 ֍, 2.5 ֍, 1 ֍, 0.5 ֍
        • Aqua annealing (17jl & 17jm): 100 ֍, 10 ֍, 5 ֍, 2.5 ֍, 1 ֍, 0.5 ֍
      • Variation: 100 V, 1 h

    Native PAGE of annealed primers (17jl & 17jm)

    Investigators: Camilla Maerz

    Aim of the experiment: M.A.X restriction system - Primer annealing test

    Procedure:

    • Native DNA PAGE standard protocol
      • tested samples:
        • HEPES annealing (17jl & 17jm): 100 ֍, 10 ֍, 5 ֍, 2.5 ֍, 1 ֍, 0.5 ֍
        • Aqua annealing (17jl & 17jm): 100 ֍, 10 ֍, 5 ֍, 2.5 ֍, 1 ֍, 0.5 ֍
      • Variation: 60 V, 2 h

    Native Page of annealed primers (17jl & 17jm; 17jh & 17ji)

    Investigators: Camilla Maerz

    Aim of the experiment: M.A.X restriction system - Primer annealing test

    Procedure:

    • Native DNA PAGE standard protocol
      • tested samples:
        • HEPES annealing (17jl & 17jm): 0.5 ֍
        • Aqua annealing (17jl & 17jm): 0.5 ֍
        • Composite Buffer annealing (17jh & 17 jm): 0.5 ֍
        • all Primers (ssDNA): 1 ֍

    2017-08-28 - 2017-09-03

    Native PAGE of annealed primers (HEPES annealing & Composite Buffer annealing) of the same concentration to test if all annealings have the same quality

    Investigators: Camilla Maerz

    Aim of the experiment: M.A.X restriction system - Primer annealing test

    Procedure:

    • all samples were diluted to a final concentration of 0.5 ֍
    • Native DNA PAGE standard protocol

    Restriction digest of control annealings (mutC)

    Investigators: Camilla Maerz

    Aim of the experiment: M.A.X restriction system

    Procedure:

    • Restriction Digest (MnlI) of aqua annealing sample: mutC (0.5 ֍)
      • 2 ֌ MnlI
      • 5 ֌ CutSmart
      • 25 ֌ mutC dsDNA
      • 18 ֌ nuclease-free H2O
      • Incubation: 37 у, 1 h
      • Inactivation: 20 min., 65 у
    • Native DNA PAGE standard protocol
      • Tested samples:
        • digested mutC sample
        • native mutC sample
        • ssDNA primers (17jl & 17jm)

    Restriction digest of aqua & HEPES annealing

    Investigators: Camilla Maerz

    Aim of the experiment: M.A.X restriction system

    Procedure:

    • Restriction Digest (MnlI) of aqua & HEPES annealing sample: mutC (0.5 ֍)
      • 2 ֌ MnlI
      • 5 ֌ CutSmart
      • 25 ֌ mutC dsDNA
      • 18 ֌ nuclease-free H2O
      • Incubation: 37 у, 1 h
      • Inactivation: 20 min., 65 у
    • Native DNA PAGE standard protocol
      • Tested samples:
        • digested mutC sample (aqua & HEPES annealing)
        • native mutC sample
        • ssDNA primers (17jl & 17jm)

    Aqua annealing of mutA, mutT, mutG and mutC

    Investigators: Camilla Maerz

    Aim of the experiment: M.A.X restriction system

    Procedure:

    • Aqua annealing standard protocol (final concentration: 0.5 ֍)
      • mutA: 17jf & 17jg
      • mutT: 17jj & 17jk
      • mutG: 17jh & 17ji
      • mutC: 17jl & 17jm
    • final volume of each sample: 50 ֌

    2017-09-04 - 2017-09-10

    Restriction digest of control annealings (mutA, mutT, mutG, mutC) and aqua annealing of oligo2

    Investigators: Camilla Maerz

    Aim of the experiment: M.A.X restriction system

    Procedure:

    • Oligo2 annealing
      • Aqua annealing standard protocol (final concentration: 0.5 ֍)
    • Restricted samples:
      • MnlI -> mutC, oligo2
      • SapI -> mutG, oligo2
      • BsaI -> mutT, oligo2
      • EciI -> mutA, oligo2
    • Restriction Digest:
      • 1 ֌ Enzyme
      • 5 ֌ CutSmart
      • 25 ֌ mutC dsDNA
      • 19 ֌ nuclease-free H2O
      • Incubation: 37 у, 1 h
      • Inactivation: 20 min., 65 у
    • Native DNA PAGE standard protocol

    2017-09-11 - 2017-09-17

    Annealing, restriction digest and native PAGE of all samples

    Investigators: Camilla Maerz

    Aim of the experiment: M.A.X restriction system

    Procedure:

    • Aqua annealing standard protocol (final concentration: 0.5 ֍)
      • mutA: 17jf & 17jg
      • mutT: 17jj & 17jk
      • mutG: 17jh & 17ji
      • mutC: 17jl & 17jm
      • oligo2: olgio2_sense & oligo2_antisense
    • final volume of each sample: 50 ֌
    • Restricted samples:
      • MnlI -> mutC, oligo2
      • SapI -> mutG, oligo2
      • BsaI -> mutT, oligo2
      • EciI -> mutA, oligo2
    • Restriction Digest:
      • 1 ֌ Enzyme
      • 5 ֌ CutSmart
      • 25 ֌ mutC dsDNA
      • 19 ֌ nuclease-free H2O
      • Incubation: 37 у, 1 h
      • Inactivation: 20 min., 65 у
    • Native DNA PAGE standard protocol

    Annealing, restriction digest and native PAGE of all samples

    Investigators: Camilla Maerz

    Aim of the experiment: M.A.X restriction system

    Procedure:

    • Aqua annealing standard protocol (final concentration: 0.5 ֍)
      • mutA: 17jf & 17jg
      • mutT: 17jj & 17jk
      • mutG: 17jh & 17ji
      • mutC: 17jl & 17jm
      • oligo2: olgio2_sense & oligo2_antisense
    • final volume of each sample: 2x50 ֌
    • Restricted samples:
      • MnlI -> mutC
      • SapI -> mutG, oligo2
      • BsaI -> mutT
      • EciI -> mutA
    • Restriction Digest:
      • 1 ֌ Enzyme
      • 5 ֌ CutSmart
      • 25 ֌ mutC dsDNA
      • 19 ֌ nuclease-free H2O
      • Incubation: 37 у, 1 h
      • Inactivation: 20 min., 65 у
    • Native DNA PAGE standard protocol

    2017-09-18 - 2017-09-24

    -Agarose gel electrophoresis (2%)

    Investigators: Agarose, gel, electrophoresis, of, digested, mutA,, mutT,, mutG, &, mutC

    Aim of the experiment: CMZ

    Procedure:

    • 60 V, 40 min

    Annealing, restriction digest and native PAGE of all samples

    Investigators: Camilla Maerz

    Aim of the experiment: M.A.X restriction system

    Procedure:

    • Aqua annealing standard protocol (final concentration: 0.5 ֍)
      • mutA: 17jf & 17jg
      • mutT: 17jj & 17jk
      • mutG: 17jh & 17ji
      • mutC: 17jl & 17jm
      • oligo2: olgio2_sense & oligo2_antisense
    • final volume of each sample: 2x50 ֌
    • Restricted samples:
      • MnlI -> mutC
      • SapI -> mutG, oligo2
      • BsaI -> mutT
      • EciI -> mutA
    • Restriction Digest:
      • 1 ֌ Enzyme
      • 5 ֌ CutSmart
      • 25 ֌ mutC dsDNA
      • 19 ֌ nuclease-free H2O
      • Incubation: 37 у, 1 h
      • Inactivation: 20 min., 65 у
    • Native DNA PAGE standard protocol

    Colony PCR of pSB3K5_mRFP_link_ccdB and pSB1C3_PtNTT2(31-575)-GFP

    Investigators: Camilla Maerz

    Aim of the experiment: Construction of pSB1C3-PlacUV5_PtNTT2

    Procedure:

    • PCR
      • Go Tag (Promega) protocol
      • Primer: VR, VF2
      • Danaturation: 56 у
      • Elongation: 90 s & 180 s
    • Agarose gel electrophoresis (1%)
      • 100 V, 30 min

    2017-09-25 - 2017-10-01

    -Aqua annealing standard protocol (final concentration: 0.5 ֍)

    Investigators: Aqua, annealing, of, mutA-1,, mutG-1,, mutC-1,, mutG-1, &, oligo1

    Aim of the experiment: ??

    Procedure:

    • mutA-1: 17iv & 17iw
      • mutT-1: 17jb & 17jc
      • mutG-1: 17ix & 17iy
      • mutC-1: 17jd & 17jc
      • oligo1: olgio1_sense & oligo1_antisense
    • final volume: 50 ֌

    2017-10-02 - 2017-10-08

    UBP-PCR with TiTaq

    Investigators: Camilla Maerz

    Aim of the experiment: PCR with UBP

    Procedure:

    • PCR
      • PCR-UBP standard protocol (TiTaq)
      • samples:
        • Ligation of: mutA, mutT, mutG, mutC and oligo 2 (1 ng/֌)
      • Primer: 17vt & 17vu
      • Primer annealing: 56 у
      • Elongation: 20 s
    • PCR and DNA purification kit (Macherey-Nagel)
    • Restriction Digest
      • 1 ֌ Enzyme
      • 5 ֌ CutSmart
      • 25 ֌ mutC dsDNA
      • 19 ֌ nuclease-free H2O
      • Incubation: 37 у, 1 h
      • Inactivation: 20 min., 65 у
    • Restricted samples:
      • MnlI -> mutC
      • SapI -> mutG
      • BsaI -> mutT
      • EciI -> mutA
      • EciI, BsaI, SapI, MnlI -> oligo2
      • Variation: oligo2 was digested with 0.5 ֌ of each enzyme
    • Agarose gel electrophoresis (2 %)
      • 100 V, 20 min

    UBP-PCR with GoTaq G2 and TiTaq

    Investigators: Camilla Maerz

    Aim of the experiment: PCR with UBP

    Procedure:

    • PCR with TiTaq:
      • PCR-UBP standard protocol (TiTaq)
      • samples:
        • Ligation of: mutA, mutT, mutG, mutC and oligo 2 (5 ng/֌)
      • Primer: 17 vt & 17 vu
      • Primer annealing: 56 у
      • Elongation: 20 s
    • PCR with GoTaq:
      • PCR-UBP standard protocol (TiTaq)
      • samples:
        • Ligation of: mutA, mutT, mutG and mutC (5 ng/֌)
      • Primer: VR & VF2
      • Primer annealing: 56 у
      • Elongation: 20 s
    • PCR with GoTaq:
      • PCR-UBP standard protocol (TiTaq)
      • samples:
        • Ligation of: mutA, mutT, mutG, mutC and oligo2 (5 ng/֌)
      • Primer: 17 vt & 17 vu
      • Primer annealing: 56 у
      • Elongation: 20 s

    Restriction digest of ligated oligo2

    Investigators: Camilla Maerz

    Aim of the experiment: PCR with UBP

    Procedure:

    • Restriction Digest
      • 5 ֌ CutSmart
      • 30 ֌ ligated oligo2 DNA
      • 2 ֌ EcoRV
      • filled up to final volume 50 ֌ with ddH20
      • Incubation: 120 min, 37 у
      • Inactivation: 20 min, 65 у

    2917-06-14 - 2917-06-20

    Duplicate the KRX-e.coli

    Investigators: Christina Drake, Denise Kerkhoff

    Aim of the experiment: Plated out: KRX-e.coli as glycostocsolution

    Procedure:

    • plated out on kanamycin
    • incubation at 37°C