Team:TU Darmstadt/labbook/Biobricks

From 2013.igem.org

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                           <td>&nbsp;</td>
                           <td>&nbsp;</td>
                           <td><ul>
                           <td><ul>
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                               <li>hier muss noch etwas hinzugefügt  werden</li>
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                               <li>Gel of Biobrick producing cells<br>
 +
M.Marker NEB Color Prestaind Broadrange<br>
 +
2.LssmOrange supernatant<br>
 +
3.mKate cells<br>
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4.Control (no visible band)<br>
 +
</li>
                               </ul></td>
                               </ul></td>
                           <td><img src="https://static.igem.org/mediawiki/2013/c/cf/Darmstadt13_biobrick_Sdspage_29_09_13.png" width="50%"  alt="xxx"></td>
                           <td><img src="https://static.igem.org/mediawiki/2013/c/cf/Darmstadt13_biobrick_Sdspage_29_09_13.png" width="50%"  alt="xxx"></td>

Revision as of 18:27, 4 October 2013

Labbook | Protocols | Materials |


01.08 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 ' assembly was successful
  
01.08 Purification  
 
  • using Wizard SV Gel and PCR Clean-Up System (Promega)
    • TLO c = 7,7 ng/µL
    • CMK c = 7,1 ng/µL
  
02.08 Isolation of LssmOrange and mKate  
 
  • PCR mixture (50 µL total volume)
    • 1.5 µl DMSO
    • 1 µL dNTPs
    • 4 µL Pfu Polymerase
    • 10 µL 5x Pfu buffer Buffer with MgSO4
    • 1 µL reverse primer
    • 1 µL forward primer
    • 5 µl template
    • add 50 µl H2O

  • template and primer specifications
    • LssmOrange: TLO (c = 7,7 ng/µL, 01.08) + LO-pre-F + LO-suf-R
    • mKate: CMK (c = 7,1 ng/µL, 01.08) + mKate-suf-R + mKate-pre-ATG-F

  • PCR program (35 cycles)
    • initial denaturation 95°C, 300s
    • denaturation 95°C, 30s
    • annealing 55°C, 55s
    • elongation 72°C, 2 min
    • final elongation 72°C, 300s

  • preparative 1% agarose gel
    • PCR batches show the expected bands ' isolation was successful
02.08 Purification  
 
  • using Wizard SV Gel and PCR Clean-Up System (Promega)
    • LssmOrange1 c = 78,5 ng/µL 260/280 = 1,87 230/260 = 1,19
    • mKate1 c = 45,8 ng/µL 260/280 = 1,78 230/260 = 0,49
    • mKate2 c = 44,9 ng/µL 260/280 = 1,89 230/260 = 0,47
  
02.08 Restriction of LssmOrange and mKate  
 
  • double digest with PstI and EcoRI for 1 hour at 37°C
  
03.08 Purification  
 
  • using Wizard SV Gel and PCR Clean-Up System (Promega)
    • LssmOrange1 c = 51,6 ng/µL 260/280 = 1,82 230/260 = 0,63
    • mKate1 c = 45,4 ng/µL 260/280 = 1,9 230/260 = 0,48
    • mKate2 c = 17,0 ng/µL 260/280 = 1,87 230/260 = 0,12
  
03.08 Ligation of pSB1C3 with LssmOrange and pSB1C3 with mKate  
 
  • ligation mixture
    • 120 ng pSB1C3 cut with EcoRI and PstI
    • 124 ng insert cut with EcoRI and PstI
    • 1 µL T4 DNA ligase (NEB)
    • 2 µL 10x t4 DNA ligase buffer (NEB)
    • add to 20 µL

  • incubation for 30 minutes at room temperature
  
03.08 Transformation  
 
  • 2 µL of every ligation batch were transformed into E.coli Top10 according to heat shock protocol
    • only few colonies grew on LB-Cam plates
  
05.08 Colony PCR of pSB1C3[LssmOrange] clones and pSB1C3[mKate] clones  
 
  • PCR mixture (50 µL total volume)
    • 1µl VR primer (10 mM)
    • 1µl VF2 primer (10 mM)
    • 5µl bacterial culture in LB-Cam medium
    • 2µl MgCl2
    • 1µl dNTP Mix
    • 1µl DMSO
    • 5µl 10x Taq Buffer
    • 1µl Taq-Polymerase
    • 33µl nucleasefree H2O

  • PCR Program
    • initial denaturation 300s 95°C
    • denaturation 30s 95°C
    • annealing 30s 55°C
    • elongation 60s 72°C
    • final elongation 300s 72°C

  • analytical 1% agarose gel
    • no colony yielded a band of approximately 800 bp, which would account for a positive result
  
09.08 Ligation of pSB1C3 with LssmOrange and pSB1C3 with mKate  
 
  • ligation mixture
    • 120 ng pSB1C3 cut with EcoRI and PstI
    • 124 ng insert cut with EcoRI and PstI
    • 1 µL T4 DNA ligase (NEB)
    • 2 µL 10x t4 DNA ligase buffer (NEB)
    • add to 20 µL
  • incubation for 2 hours at 37°C and additionally incubation over 24 hours at 8°C
 
10.08 Transformation  
 
  • 2 µL of every ligation batch were transformed into E.coli Top10 according to heat shock protocol
    • a lot of colonies grew on LB-Cam plates
  
12.08 Colony PCR of pSB1C3[LssmOrange] clones and pSB1C3[mKate] clones  
 
  • PCR mixture (50 µL total volume)
    • 1µl VR primer (10 mM)
    • 1µl VF2 primer (10 mM)
    • 5µl bacterial culture in LB-Cam medium
    • 2µl MgCl2
    • 1µl dNTP Mix
    • 1µl DMSO
    • 5µl 10x Taq Buffer
    • 1µl Taq-Polymerase
    • 33µl nucleasefree H2O

  • PCR Program
    • initial denaturation 300s 95°C
    • denaturation 30s 95°C
    • annealing 30s 55°C
    • elongation 60s 72°C
    • final elongation 300s 72°C

  • analytical 1% agarose gel
    • a lot clones gave a positive result
  
13.08 Sequencing of pSB1C3[LssmOrange] clones and pSB1C3[mKate] clones  
 
  • results show that all analyzed clones contain the expected sequence
  • one pSB1C3[LssmOrange] clone and one pSB1C3[mKate] clone were chosen
 
26.08 Plasmid prep of pSB1C3[LssmOrange] clones and pSB1C3[mKate] clones  
 
  • using Wizard SV Gel and PCR Clean-Up System (Promega)
    • pSB1C3[LssmOrange] c = 96,4 ng/µL 260/280 = 1,68 230/260 = 1,46
    • pSB1C3[mKate] c = 74,8 ng/µL 260/280 = 1,86 230/260 = 1,82
  
13.09 Inoculation of DH5? pPR-IBA2 and subsequent plasmid prep  
 
  • 6 mL LB-Amp were inoculated with 50 µL of DH5? pPR-IBA2
    • using Wizard SV Gel and PCR Clean-Up System (Promega)
    • pPR-IBA2 c = 71,6 ng/µL 260/280 = 1,82 260/230 = 1,76
  
13.09 Isolation PCR of LssmOrange and mKate  
 
  • PCR mixture (50 µL total volume)
    • 5x Q5 Reaction Buffer
    • 5x Q5 GC Enhancer
    • 1 µL dNTPs (10mM)
    • 2,5 µL forward primer (10 mM)
    • 2,5 µL reverse primer (10 mM)
    • 1 µL template
    • 0,5 µL Q5 Polymerase
    • 22,5 µL nucleasefree water

  • template and primer specifications
    • LssmOrange: pSB1C3[LssmOrange ] (c = 96,4 ng/µL, 1:100 dilution) + lssmorange pprf + lssmorange ppr
    • mKate: pSB1C3[mKate] (c = 74,8 ng/µL, 1:80 dilution) + mkate pprf + mkate ppr

  • PCR program (35 cycles)
    • initial denaturation, 60 s, 98°C
    • denaturation, 10 s, 98°C
    • annealing, 30 s, 65°C
    • elongation, 60 s, 72°C
    • final elongation, 120 s, 72°C

  • analytical 1% agarose gel
    • each PCR batch displays the expected band of approximately 700 bp
  
13.09 Purification  
 
  • using Wizard SV Gel and PCR Clean-Up System (Promega)
    • LssmOrange1 c = 51,4 ng/µL 260/280 = 1,69 230/260 = 1,59
    • LssmOrange1 c = 51,6 ng/µL 260/280 = 1,66 230/260 = 1,55
    • mKate1 c = 38,4 ng/µL 260/280 = 1,64 230/260 = 1,43
    • mKate2 c = 37,8 ng/µL 260/280 = 1,59 230/260 = 1,42
  
13.09 Restriction of pPR-IBA2, LssmOrange and mKate  
 
  • double digest with NheI and PstI at 37°C for 4 hours

  • preparative 1% agarose gel
    • LssmOrange and mKate show a band of the expected size
    • pPR-IBA2 shows intense band at 3000bp, weak band at 2250bp and weak band below 100bp
      • we were unsure which band the correct one is, and cut out the 3000band as well as 2250band
  
14.09 Purification of pPR-IBA2, LssmOrange and mKate  
 
  • using Wizard SV Gel and PCR Clean-Up System (Promega)
    • pPR-IBA2 (3000band) c = 31,2 ng/µL 260/280 = 1,88 230/260 = 1,00
    • pPR-IBA2 (2250band) c = 9,5 ng/µL 260/280 = 1,39 230/260 = 0,38 (neglected due to low purity)
    • LssmOrange c = 57,3 ng/µL 260/280 = 1,74 230/260 = 1,53
    • mKate c = 22,0 ng/µL 260/280 = 2,04 230/260 = 1,16
  
14.09 Ligation  
 
  • ligation batch of pPR-IBA2[LssmOrange]
    • 3 µL pPR-IBA2 3000 (93 ng)
    • 1,5 µL LssmOrange (72 ng)
    • 2 µL 10x T4 Ligase Buffer (NEB)
    • 1 µL T4 Ligase (NEB)
    • 12,5 µL nucleasefree water

  • ligation batch of pPR-IBA2[mKate]
    • 3 µL pPR-IBA2 3000 (93 ng)
    • 3,5 µL mKate (72 ng)
    • 2 µL 10x T4 Ligase Buffer (NEB)
    • 1 µL T4 Ligase (NEB)
    • 10,5 µL nucleasefree water

  • incubation at room temperature for 30 minutes
  
15.09 Transformation of pPR-IBA2[LssmOrange] and pPR-IBA2[mKate]  
 
  • 2 µL of ligation batch were transformed into E.coli DH5? according to heat shock protocol
    • a lot of colonies grew on LB-Amp plates
  
16.09 Colony PCR of pPR-IBA2[LssmOrange] and pPR-IBA2[mKate]  
 
  • PCR mixture (50 µL total volume)
    • 1 µL forward primer
    • 1 µL reverse primer
    • colony
    • 2 µl MgCl2
    • 1 µl dNTP Mix
    • 1 µl DMSO
    • 5 µl 10x Taq buffer
    • 1 µl Taq-Polymerase
    • 38 µl H2O

  • template and primer specifications
    • pSB1C3[LssmOrange]: lssmorange pprf + lssmorange ppr
    • pSB1C3[mKate]: mkate pprf + mkate ppr

  • PCR Program
    • initial Denaturation 300s 95°C
    • Denaturation 30s 95°C
    • Annealing 30s 65°C
    • Elongation 60s 72°C
    • Final Elongation 300s 72°C

  • analytical 1% agarose gel
    • 2 positive clones for pPR-IBA2-LssmOrange
    • 7 positive clones for pPR-IBA2-mKate
 xxx
19.09 Plasmid prep  
 
  • using Wizard SV Gel and PCR Clean-Up System (Promega)
    • pPR-IBA2[LssmOrange] clone 5 c = 51,2 ng/µL
    • pPR-IBA2[LssmOrange] clone 10 c = 55,5 ng/µL
    • pPR-IBA2[mKate] clone 2 c = 32,3 ng/µL
    • pPR-IBA2[mKate] clone 3 c = 35,2 ng/µL
    • pPR-IBA2[mKate] clone 4 c = 29,1 ng/µL
    • pPR-IBA2[mKate] clone 8 c = 37,6 ng/µL
  
19.09 Transformation in BL21DE3  
 
  • 1 µL of plasmid was transformed into E.coli DH5? according to heat shock protocol
    • biofilm grew on LB-Amp plates
 
21.09 Induction with IPTG  
 
  • pPR-IBA2[LssmOrange] and pPR-IBA2[mKate] clones were cultivated in SOC-Amp medium at 37°C and 150 rpm to an OD600 = 0,6
  • subsequent induction with IPTG (2 mM end concentration) and cultivation overnight at 30°C and 150 rpm
    • induction worked for pPR-IBA2[LssmOrange] clone 10 and pPR-IBA2[mKate] clone 3 and clone 4
  
23.09 Spectral analysis of induced clones  
 
  • cells and cell lysate was analyzed using an fluorospectrometer
  
27.09 SDS-PAGE  
 
  • Gel of Biobrick producing cells
    M.Marker NEB Color Prestaind Broadrange
    2.LssmOrange supernatant
    3.mKate cells
    4.Control (no visible band)
 xxx

Retrieved from "http://2013.igem.org/Team:TU_Darmstadt/labbook/Biobricks"