Difference between revisions of "Team:Dundee/labjournal manu"

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                   under data-target refer to a modal specifically made for each image under image modals section-->
 
                   under data-target refer to a modal specifically made for each image under image modals section-->
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              </div>
 
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              <div id="collapsiblew5-5" class="collapse box-content">
 
  
  
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         <div class="modal-title" id="myModalLabel">Plating Protocol</div>
 
         <div class="modal-title" id="myModalLabel">Plating Protocol</div>
 
       </div>
 
       </div>
−
       <div class="modal-body"><p>
+
       <div class="modal-body"> <!-- <p>?? -->
−
    1. Agar plates with required antibiotic were taken out of 4<sup>o</sup>C to warm to room temperature.
+
      <ol>
−
</p>
+
        <li>Agar plates with required antibiotic were taken out of 4<sup>o</sup>C to warm to room temperature.</li>
−
<p>
+
        <li>Cells or colonies were picked up from the source with a toothpick under sterile conditions.</li>
−
    2. Cells or colonies were picked up from the source with a toothpick under sterile conditions.
+
        <li>Fresh agar plates were inoculated with cells on the toothpick.</li>
−
</p>
+
        <li>Agar plates were incubated at 4<sup>o</sup>C over night.</li>
−
<p>
+
      </ol> <!-- </p>?? -->
−
    3. Fresh agar plates were inoculated with cells on the toothpick.
+
−
</p>
+
−
<p>
+
−
    4. Agar plates were incubated at 4<sup>o</sup>C over night.
+
−
</p>
+
 
       </div>
 
       </div>
 
       <div class="modal-footer">
 
       <div class="modal-footer">
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       </div>
 
       </div>
 
       <div class="modal-body"><p>
 
       <div class="modal-body"><p>
−
    1. Agar plates with required antibiotic were taken out of 4<sup>o</sup>C to warm to room temperature.
+
 
−
</p>
+
      <ol>
−
<p>
+
        <li>Agar plates with required antibiotic were taken out of 4<sup>o</sup>C to warm to room temperature.</li>
−
    2. Cells or colonies were picked up from the source with a toothpick under sterile conditions.
+
        <li>Cells or colonies were picked up from the source with a toothpick under sterile conditions.</li>
−
</p>
+
        <li>5ml of LB medium, supplemented with the required antibiotic, were inoculated with the toothpick.</li>
−
<p>
+
        <li>Tubes were incubated at 37<sup>o</sup>C in a rotary incubator at 200rpm over night.</li>
−
    3. 5ml of LB medium, supplemented with the required antibiotic, were inoculated with the toothpick.
+
      </ol>
−
</p>
+
−
<p>
+
−
    4. Tubes were incubated at 37<sup>o</sup>C in a rotary incubator at 200rpm over night.
+
−
</p>
+
 
       </div>
 
       </div>
 
       <div class="modal-footer">
 
       <div class="modal-footer">
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         <div class="modal-title" id="myModalLabel">Miniprep Protocol</div>
 
         <div class="modal-title" id="myModalLabel">Miniprep Protocol</div>
 
       </div>
 
       </div>
−
       <div class="modal-body"><p>
+
       <div class="modal-body">
−
    1. 5ml bacterial overnight culture was pelleted by stepwise centrifugation in 1.5ml microcentrifuge tubes at 13300rpm for 3 minutes at room temperature.
+
 
−
</p>
+
      <ol>
−
<p>
+
        <li>5ml bacterial overnight culture was pelleted by stepwise centrifugation in 1.5ml microcentrifuge tubes at 13300rpm for 3 minutes at room temperature.</li>
−
    2. Pelleted bacterial cells were resuspended in 250µl of Buffer P1.
+
        <li>Pelleted bacterial cells were resuspended in 250µl of Buffer P1.</li>
−
</p>
+
        <li>250µl of Buffer P2 was added and mixed thoroughly by inverting the tube 4-6 times until solution became clear.</li>
−
<p>
+
        <li>350µl of Buffer N3 was added and immediately and thoroughly mixed by inverting the tube 4-6 times.</li>
−
    3. 250µl of Buffer P2 was added and mixed thoroughly by inverting the tube 4-6 times until solution became clear.
+
        <li>The mixture was centrifuged for 10 minutes at 13300rpm in a microcentrifuge.</li>
−
</p>
+
        <li>The supernatant was applied to a QIAprep spin column by pipetting.</li>
−
<p>
+
        <li>500µl of Buffer PB was added to the QIAprep spin column and centrifuged for 1 minute. The flow-through was discarded.</li>
−
    4. 350µl of Buffer N3 was added and immediately and thoroughly mixed by inverting the tube 4-6 times.
+
        <li>750µl of Buffer PE was added to the QIAprep spin column and centrifuged for 1 minute. The flow-through was discarded.</li>
−
</p>
+
        <li>The QIAprep spin column was spun for an additional minute to remove residual wash buffer.</li>
−
<p>
+
        <li>The QIAprep spin column was placed in a clean 1.5ml microcentrifuge and the DNA eluted by adding 30µl of H<sub>2</sub>O to the QIAprep spin column and centrifuging for 1 minute. </li>
−
    5. The mixture was centrifuged for 10 minutes at 13300rpm in a microcentrifuge.
+
      </ol>
−
</p>
+
      <!-- </div>?? -->
−
<p>
+
−
    6. The supernatant was applied to a QIAprep spin column by pipetting.
+
−
</p>
+
−
<p>
+
−
    7. 500µl of Buffer PB was added to the QIAprep spin column and centrifuged for 1 minute. The flow-through was discarded.
+
−
</p>
+
−
<p>
+
−
    8. 750µl of Buffer PE was added to the QIAprep spin column and centrifuged for 1 minute. The flow-through was discarded.  
+
−
</p>
+
−
<p>
+
−
    9. The QIAprep spin column was spun for an additional minute to remove residual wash buffer.
+
−
</p>
+
−
<p>
+
−
    10. The QIAprep spin column was placed in a clean 1.5ml microcentrifuge and the DNA eluted by adding 30µl of H2O to the QIAprep spin column and centrifuging for 1 minute.  
+
−
</p>
+
−
</div>
+
 
       </div>
 
       </div>
 
       <div class="modal-footer">
 
       <div class="modal-footer">
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         <div class="modal-title" id="myModalLabel">Gel Extraction Protocol</div>
 
         <div class="modal-title" id="myModalLabel">Gel Extraction Protocol</div>
 
       </div>
 
       </div>
−
       <div class="modal-body"><p>
+
       <div class="modal-body">
−
    1. The desired DNA fragment was excised from the agarose gel with a clean, sharp scalpel.
+
 
−
</p>
+
      <ol>
−
<p>
+
        <li>The desired DNA fragment was excised from the agarose gel with a clean, sharp scalpel.</li>
−
    2. The gel slice was transferred into a microcentrifuge tube. 700µl of Buffer QG was added.
+
        <li>The gel slice was transferred into a microcentrifuge tube. 700µl of Buffer QG was added.</li>
−
</p>
+
        <li>The gel slice was incubated in a water bath at 50°C for 10 min (or until the gel slice has completely dissolved). The tube was vortexed every 2–3 min to help dissolve gel.</li>
−
<p>
+
        <li>After the gel slice had been dissolved completely, the color of the mixture was checked that it was yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture was orange or violet, 10 μl 3 M sodium acetate, pH 5.0 would be added, and mixed. The color of the mixture would turn yellow.</li>
−
    3. The gel slice was incubated in a water bath at 50°C for 10 min (or until the gel slice has completely dissolved). The tube was vortexed every 2–3 min to help dissolve gel.
+
        <li>230µl of isopropanol was added to the sample and mixed.</li>
−
</p>
+
        <li>A QIAquick spin column was placed in a 2ml collection tube. The sample was applied to the QIAquick spin column and centrifuged for 1 minute. The flow-through was discarded.</li>
−
<p>
+
        <li>500µl of QG buffer was added to the QIAquick spin column and centrifuged for 1 minute. The flow-through was discarded.</li>
−
    4. After the gel slice had been dissolved completely, the color of the mixture was checked that it was yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture was orange or violet, 10 μl 3 M sodium acetate, pH 5.0 would be added, and mixed. The color of the mixture would turn yellow.
+
        <li>750µl of Buffer PE was added to the QIAquick spin column and centrifuged for 1 minute. The flow-through was discarded.</li>
−
</p>
+
        <li>The QIAprep spin column was spun for an additional minute to remove residual wash buffer.</li>
−
<p>
+
        <li>The QIAprep spin column was placed in a clean 1.5ml microcentrifuge and the DNA eluted by adding 30µl of H2O to the QIAprep spin column and centrifuging for 1 minute.</li>
−
    5. 230µl of isopropanol was added to the sample and mixed.
+
      </ol>
−
</p>
+
 
−
<p>
+
−
    6. A QIAquick spin column was placed in a 2ml collection tube. The sample was applied to the QIAquick spin column and centrifuged for 1 minute. The flow-through was discarded.
+
−
</p>
+
−
<p>
+
−
    7. 500µl of QG buffer was added to the QIAquick spin column and centrifuged for 1 minute. The flow-through was discarded.
+
−
</p>
+
−
<p>
+
−
    8. 750µl of Buffer PE was added to the QIAquick spin column and centrifuged for 1 minute. The flow-through was discarded.  
+
−
</p>
+
−
<p>
+
−
    9. The QIAprep spin column was spun for an additional minute to remove residual wash buffer.
+
−
</p>
+
−
<p>
+
−
    10. The QIAprep spin column was placed in a clean 1.5ml microcentrifuge and the DNA eluted by adding 30µl of H2O to the QIAprep spin column and centrifuging for 1 minute.  
+
−
</p>
+
−
</div>
+
 
       </div>
 
       </div>
 
       <div class="modal-footer">
 
       <div class="modal-footer">
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         <div class="modal-title" id="myModalLabel">Transformation Protocol</div>
 
         <div class="modal-title" id="myModalLabel">Transformation Protocol</div>
 
       </div>
 
       </div>
−
       <div class="modal-body"><p>
+
       <div class="modal-body">
−
    1. 100µl of competent cells were defrosted. Thaw on ice.
+
 
−
</p>
+
      <ol>
−
<p>
+
        <li>100µl of competent cells were defrosted. Thaw on ice.</li>
−
    2. Agar plates were taken out of 4<sup>o</sup>C to warm to room temperature.
+
        <li>Agar plates were taken out of 4<sup>o</sup>C to warm to room temperature.</li>
−
</p>
+
        <li>1-5µl of DNA was added to cells.</li>
−
<p>
+
        <li>Cell and DNA mixture was left on ice for 20 minutes.</li>
−
    3. 1-5µl of DNA was added to cells.
+
        <li>Mixture was placed in 42<sup>o</sup>C water bath for 90 seconds.</li>
−
</p>
+
        <li>Mixture was put back on ice for 2 minutes.</li>
−
<p>
+
        <li>1ml of LB (without antibiotic) was added to cells.</li>
−
    4. Cell and DNA mixture was left on ice for 20 minutes.
+
        <li>Cells were allowed to grow for 1 hour at 37<sup>o</sup>C in a shaking incubator.</li>
−
</p>
+
        <li>Cells were then spun down and the supernatant discarded.</li>
−
<p>
+
        <li>The pellet was resuspended in the 100µl.</li>
−
    5. Mixture was placed in 42<sup>o</sup>C water bath for 90 seconds.
+
        <li>Cells were plated on agar plates with appropriate antibiotic.</li>
−
</p>
+
      </ol>
−
<p>
+
 
−
    6. Mixture was put back on ice for 2 minutes.
+
−
</p>
+
−
<p>
+
−
    7. 1ml of LB (without antibiotic) was added to cells.
+
−
</p>
+
−
<p>
+
−
    8. Cells were allowed to grow for 1 hour at 37<sup>o</sup>C in a shaking incubator.
+
−
</p>
+
−
<p>
+
−
    9. Cells were then spun down and the supernatant discarded.
+
−
</p>
+
−
<p>
+
−
    10. The pellet was resuspended in the 100µl.
+
−
</p>
+
−
<p>
+
−
    Cells were plated on agar plates with appropriate antibiotic.
+
−
</p>
+
−
</div>
+
 
       </div>
 
       </div>
 
       <div class="modal-footer">
 
       <div class="modal-footer">
Line 3,924: Line 3,862:
 
     <strong><u>SDS-PAGE and Western Blotting </u></strong>
 
     <strong><u>SDS-PAGE and Western Blotting </u></strong>
 
</p>
 
</p>
−
<p>
+
 
−
    1. 50µl of an overnight culture was used to inoculate 5ml of fresh LB containing the appropriate antibiotics and left to grow at 37<sup>o</sup>C in a
+
      <ol>
−
    shaking incubator.
+
        <li>50µl of an overnight culture was used to inoculate 5ml of fresh LB containing the appropriate antibiotics and left to grow at 37<sup>o</sup>C in a shaking incubator.</li>
−
</p>
+
        <li>Once the OD<sub>600</sub>=0.6-0.8, 1mM of IPTG was added to the culture and left to grow for a further 3 hours.</li>
−
<p>
+
        <li>1ml of each culture was taken and spun down in a microcentrifuge for 3 minutes.</li>
−
    2. Once the OD<sub>600</sub>=0.6-0.8, 1mM of IPTG was added to the culture and left to grow for a further 3 hours.
+
        <li>Meanwhile, 950µl of 2x Laemmli sample buffer was added to 50µl of B-mercaptoethanol.</li>
−
</p>
+
        <li>The supernatant was discarded from the samples and the pellet resuspended in 200µl of the above solution.</li>
−
<p>
+
        <li>The samples were then boiled at 90<sup>o</sup>C for 10 minutes.</li>
−
    3. 1ml of each culture was taken and spun down in a microcentrifuge for 3 minutes.
+
        <li>Afterwards, they were spun down for 1 minute in a microcentrifuge.</li>
−
</p>
+
        <li>At this stage, the samples could be stored at -20<sup>o</sup>C or ran on a gel immediately.</li>
−
<p>
+
        <li>Two gradient gels consisting of a 12% separating gel and a stacking gel were prepared and the samples ran at 100V until proteins reach end of the stacking gel at which point the voltage was increased to 200V for 20 minutes.</li>
−
    4. Meanwhile, 950µl of 2x Laemmli sample buffer was added to 50µl of B-mercaptoethanol.
+
        <li>One gel was stained using Instant Blue and the other was used to transfer the proteins onto a membrane.</li>
−
</p>
+
        <li>After the proteins had been transferred onto the membrane, the membrane was blocked in 5% Marvel milk overnight at 4<sup>o</sup>C.</li>
−
<p>
+
        <li>The next day, the membrane was washed 3 times in 1xTBST buffer and then suspended in 10ml of 1xTBS buffer to which the primary anti-His antibody was added and left for one hour. The protein ladder was cut off before adding the primary antibody.</li>
−
    5. The supernatant was discarded from the samples and the pellet resuspended in 200µl of the above solution.
+
        <li>The membrane was then washed 3 times in 1xTBST buffer for 5 minutes and then suspended in 10ml of 1xTBS buffer, to which the secondary antibody was added and left for one hour.</li>
−
</p>
+
        <li>The membrane was then washed in 3x10ml 1xTBST buffer for 5 minutes.</li>
−
<p>
+
        <li>The blot was then soaked in developing solution for 5 minutes before being wrapped in a plastic cover aligned with the protein ladder that was cut off at step 12.</li>
−
    6. The samples were then boiled at 90<sup>o</sup>C for 10 minutes.
+
        <li>Once in the developing room, the film was exposed to the blot for 30 seconds, 2 minutes and 5 minutes and then placed in the film developer.</li>
−
</p>
+
      </ol>
−
<p>
+
−
    7. Afterwards, they were spun down for 1 minute in a microcentrifuge.
+
−
</p>
+
−
<p>
+
−
    8. At this stage, the samples could be stored at -20<sup>o</sup>C or ran on a gel immediately.
+
−
</p>
+
−
<p>
+
−
    9. Two gradient gels consisting of a 12% separating gel and a stacking gel were prepared and the samples ran at 100V until proteins reach end of the
+
−
    stacking gel at which point the voltage was increased to 200V for 20 minutes.
+
−
</p>
+
−
<p>
+
−
    10. One gel was stained using Instant Blue and the other was used to transfer the proteins onto a membrane.
+
−
</p>
+
−
<p>
+
−
    11. After the proteins had been transferred onto the membrane, the membrane was blocked in 5% Marvel milk overnight at 4<sup>o</sup>C.
+
−
</p>
+
−
<p>
+
−
    12. The next day, the membrane was washed 3 times in 1xTBST buffer and then suspended in 10ml of 1xTBS buffer to which the primary anti-His antibody was
+
−
    added and left for one hour. The protein ladder was cut off before adding the primary antibody.
+
−
</p>
+
−
<p>
+
−
    13. The membrane was then washed 3 times in 1xTBST buffer for 5 minutes and then suspended in 10ml of 1xTBS buffer, to which the secondary antibody was
+
−
    added and left for one hour.
+
−
</p>
+
−
<p>
+
−
    14. The membrane was then washed in 3x10ml 1xTBST buffer for 5 minutes.
+
−
</p>
+
−
<p>
+
−
    15. The blot was then soaked in developing solution for 5 minutes before being wrapped in a plastic cover aligned with the protein ladder that was cut off
+
−
    at step 12.
+
−
</p>
+
−
<p>
+
−
    Once in the developing room, the film was exposed to the blot for 30 seconds, 2 minutes and 5 minutes and then placed in the film developer.
+
−
</p>
+
  
 
       </div>
 
       </div>

Revision as of 19:04, 10 August 2015

LABJOURNAL

BioSpray

Our forensic toolkit aims to use synthetic biology approaches to improve on the current methods used by crime scene investigators.

Chromium Detector

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Curabitur a tincidunt elit. Aliquam porta nibh at enim luctus, auctor consequat dolor vehicula.

Fingerprint Aging

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Curabitur a tincidunt elit. Aliquam porta nibh at enim luctus, auctor consequat dolor vehicula.

22/06 - 28/06

Summary

This week was all about getting started with the newly arrived biobrick BBa_K1058008

Day 1

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 BBa_K1058008 and confirmation of size and sequence.

Day 2

Aim of experiment: To purify BBa_K1058008 for sequence confirmation.

Protocols Used:

Click here to see our miniprep protocol.

Click here to see our sequencing protocol.

Results Miniprep:

Sample Name

Concentration

Unit

Blank

-

-

BBa_K1058008

190.93

ng/µl

Results Sequencing: Figure 1

Next Steps: PCR of parts of BBa_K1058008.

29/06 - 05/07

Summary

During this week, BBa_K1058008 was disassembled into its constituent parts, pChr, and ChrB.

Day 1

Aim of experiment: To break down BBa_K1058008 into the promoter region pChr, and the repressor gene, ChrB, while adding standard prefix and suffix to each. Furthermore optimisation of first 15 codons of ChrB in order to increase the overall rate of transcription. Restriction digest of the respective ends and subsequent ligation of pChr and ChrB into pSB1C3 for submission to registry.

Protocols Used:

Click here to see our PCR protocol.

Click here to see our gel extraction protocol.

Click here to see our restriction digest protocol.

Results:

Figure 2: Agarose gel after PCR of pChr and ChrB.

As can be seen on the image of the gel, PCR of ChrB was not successful. pChr was further processed by restriction digest with EcoRI and PstI

Next Steps: Repeat of PCR of ChrB. Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

06/07 - 12/07

Summary

During this week ligations of pChr and ChrB into pSB1C3 were finalised and the constituent parts of our newly designed chromate sensing system were prepared for ligation and transformation.

Day 1

Aim of experiment: To repeat PCR of ChrB for subsequent ligation into pSB1C3. Ligation of pChr and ChrB into pSB1C3. Transformation of each into a non-pathogenic lab strain of E. coli.

Overnight culture of strain, containing GFPmut2, as a fluorescent reporter for our Chromate sensing system

Protocols Used:

Click here to see our PCR protocol.

The annealing temperature was lowered in order to increase the chances of accomodating a primer that is not 100% complementary. Furthermore different concentrations of DMSO were tested.

Click here to see our gel extraction protocol.

Click here to see our restriction digest protocol.

Click here to see our ligation protocol. Ligations were made in 2:1 and 3:1 ratio.

Click here to see our Overnight culture protocol.

Results:

Figure 3: Agarose gel after PCR of ChrB.

2 bands were extracted from the gel for further characterisation, the strongest band, hence called ChrBs, and the band directly above, hence called ChrBw.

pChr, ChrBs, and ChrBw were ligated into pSB1C3.

pChr-pSB1C3, ChrBs-pSB1C3, and ChrBw-pSB1C3 were transformed into E. coli MC1061.

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 2

Aim of experiment: Purification of GFPmut2 from overnight cultures. Overnight cultures of recombinant MC1061, containing pChr-pSB1C3, ChrBs-pSB1C3, and ChrBw-pSB1C3 respectively. Overnight culture of pUniprom for subsequent ligation of ChrB

Protocols Used:

Miniprep for plasmid purification of overnight culture of GFPmut2.

PCR for amplification of GFPmut2 and ChrB.

Gel extraction of PCR product pf GFPmut2.

Overnight culture of MC1061 containing pChr-pSB1C3, ChrBs-pSB1C3, and ChrBw-pSB1C3 respectively. Furthermore of a strain containing the vector pUniprom.

Results: Figure 5 Since amplification of ChrB was unsuccessful, it will be repeated.

Next Steps: Sequence and size confirmation of pChr-pSB1C3, ChrBs-pSB1C3, and ChrBw-pSB1C3.

Day 3

Aim of experiment: To purify overnight cultures for size and sequence confirmation. Repeat of PCR amplification of pChr and ChrB. Restriction digestes of all parts in order to produce compatible sticky ends.

Protocols Used:

Miniprep of pChr-pSB1C3, ChrBs-pSB1C3, ChrBw-pSB1C3 and pUniprom.

Results Miniprep:

Sample Name

Concentration

Unit

Blank

-

-

pUniprom1

55.65

ng/µl

pUniprom2

64.20

ng/µl

pChr1

116.58

ng/µl

pChr2

110.13

ng/µl

pChr3

114.65

ng/µl

pChr4

105.63

ng/µl

ChrBs1

151.18

ng/µl

ChrBs2

108.37

ng/µl

ChrBs3

143.13

ng/µl

ChrBs4

183.27

ng/µl

ChrBw1

116.26

ng/µl

ChrBw2

236.99

ng/µl

ChrBw3

219.21

ng/µl

ChrBw4

208.27

ng/µl

Protocols Used:

PCR for amplification of pChr and ChrB.

Restriction digest for confirmation of size of pChr and ChrB.

Results: Figure 6 Agarose gel for size confirmation.

Protocols Used:

Sequencing of pChr and ChrB that showed expectes sizes.

Results: Figure 7 Confirmed sequence of pChr1.

Results: Figure 8 Confirmed sequence of ChrBw4.

Protocols Used:

Restriction digest of gel purified PCR-products of pChr, ChrB, and GFPmut2, using EcoRI/SpeI, BamHI/HindIII, and XbaI/PstI respecitvely.

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 4

Aim of experiment: To prepare pUniprom for insertion of ChrB.

Protocols Used:

Restriction digest of pUniprom with BamHI and HindIII.

Gel extraction of digested pUniprom

Results: Figure 9

Next Steps: Ligation of ChrB into pUniprom.

Aim of experiment: To amplify ChrB version with first 15 codons optimised for insertion into pUniprom.

Protocols Used:

PCR of optimised ChrB from the sequence confirmed ChrBw4-pSB1C3, adding restriction sites compatible to pUniprom.

Gel extraction of optimised ChrBafter PCR.

Results: Figure 10

Next Steps: Ligation of ChrB and codon optimised ChrB into pUniprom.

Day 5

Aim of experiment: Ligation of ChrB and optimised ChrB into pUniprom. Ligation of pChr and GFPmut2 into pSB1C3

Protocols Used:

Ligation of ChrB and optimised ChrB into pUniprom via BamHI and HindIII restriction sites. Ligation of pChr to GFPmut2 via Spe/Xba link and ligation of pChrGFP construct into pSB1C3 EcoRI/PstI restriction sites.

Results: N/A

Next Steps:Transformation of abovementioned ligations.

13/07 - 19/07

Summary

During this week successful transformations were identified and propagated.

Day 1

Aim of experiment: Transform recombinant vectors into E. coli chassis.

Protocols Used:

Transformation of ChrB-pUniprom, optimised ChrB-pUniprom and pChr-GFPmut2-pSB1C3 into JM110.

Results: Figure 11

Aim of experiment: Backup of digested pSB1C3 for the case that a ligation with 2 inserts does not work.

Protocols Used:

Restriction digest of pSB1C3 with EcoRI/SpeI for subsequent insertion of pChr.

Results:N/A

Next Steps: Store digested plasmid at -20oC.

Day 2

Aim of experiment: Purification of recombinant vectors for sequence and size confirmation.

Protocols Used:

Overnight culture of JM110 with ChrB-pUniprom, optimised ChrB-pUniprom and pChr-GFPmut2-pSB1C3.

Patch plating of the same colonies.

Results: N/A

Next Steps: Purification of the recombinant plasmids for confirmation of size and sequence.

Day 3

Aim of experiment: Purification of recombinant plasmids for confirmation of size and sequence.

Protocols Used:

Miniprep of ChrB-pUniprom, optimised ChrB-pUniprom and pChr-GFPmut2-pSB1C3.

Results:

Sample Name

Concentration

Unit

Blank

-

-

pSB1C3-pChr-GFPmut2_1

382.32

ng/µl

pSB1C3-pChr-GFPmut2_2

355.31

ng/µl

pSB1C3-pChr-GFPmut2_3

209.85

ng/µl

pSB1C3-pChr-GFPmut2_4

217.57

ng/µl

pUniprom-ChrB-opt_1

150.51

ng/µl

pUniprom-ChrB-opt_2

214.17

ng/µl

pUniprom-ChrB-opt_3

140.85

ng/µl

pUniprom-ChrB-opt_4

115.85

ng/µl

pUniprom-ChrB_1

134.72

ng/µl

pUniprom-ChrB_2

111.41

ng/µl

pUniprom-ChrB_3

111.31

ng/µl

pUniprom-ChrB_4

138.9

ng/µl

Protocols Used:

Restriction digest of ChrB-pUniprom and optimised ChrB-pUniprom with BamHI and HindIII. Restriction digest of pChr-GFPmut2-pSB1C3 with EcoRI/PstI.

Results: Figure 1

Protocols Used:

Colony PCR of pUniprom-ChrB-opt for elucidation of insert size or presence of insert.

Results: Figure 13 Gel after colony PCR. Colonies 2, 4, 9, and 12 were selected for sequencing.

Protocols Used:

Sequencing of pSB1C3-pChr-GFP.

Results: Figure 14 Sequencing result of pSB1C3-pChr-GFPmut2_1. The Result for pSB1C3-pChr-GFPmut2_2 was not as expected, albeit in the correct orientation.

Next Steps: Confirmation of insert size and sequence of ChrB-opt.

Day 4

Aim of experiment: To prepare ChrB-opt colonies 2, 4, 9, and 12 for sequencing.

Protocols Used:

Click here to see our overnight culture protocol.

Results:N/A

Next Steps: Purification of recombinant plasmids.

Day 5

Aim of experiment: To purify recombinant plasmids for sequence confirmation.

Protocols Used:

Miniprep of ChrB-opt 2, 4, 9, 12.

Results:

Sample Name

Concentration

Unit

Blank

-

-

pUniprom-ChrB-opt_2

255.35

ng/µl

pUniprom-ChrB-opt_4

146.77

ng/µl

pUniprom-ChrB-opt_9

211.91

ng/µl

pUniprom-ChrB-opt_12

181.08

ng/µl

Protocols Used:

Sequencing of ChrB-opt 2, 4, 9, 12.

Results: ChrB showed HindIII restriction sites and was hence cleaved into partial inserts.

Next Steps:Use alternative cloning protocol for ChrB and ChrBopt.

20/07 - 26/07

Summary

During this week an alternative cloning strategy for ChrB and ChrB-opt was employed. Furthermore, the pChr-GFP construct was cloned again in a stepwise fashion in order to clone it in the correct orientation.

Day 1 - 20/07

Aim of experiment:To compare colonies on the patch plate of pSB1C3-pChr-GFP by differentially visualising colonies that do or do not express GFP under UV light.

Results:Figure 15 pSB1C3-pChr-GFP in JM110.

Next Steps:Size confirmation and sequencing of newly selected colonies.

Day 2 - 21/07

Aim of experiment:Purification of newly selected colonies containing pSB1C3-pChr-GFP for subsequent sequence confirmation.

Protocols Used:

Miniprep for purification of pSB1C3-pChr-GFP.

Results:

Sample Name

Concentration

Unit

Blank

-

-

pSB1C3-pChr-GFP_1

313.63

ng/µl

pSB1C3-pChr-GFP_5

673.70

ng/µl

pSB1C3-pChr-GFP_6

678.02

ng/µl

pSB1C3-pChr-GFP_7

537.87

ng/µl

Next Steps:Sequencing of pSB1C3-pChr-GFP samples 1, 5, 6, and 7.

Protocols Used:

Sequencing of pSB1C3-pChr-GFP samples 1, 5, 6, and 7.

Results: Figure 16 Alignment of sequencing results.

Next Steps:Stepwise ligation of pChr-GFP into pSB1C3.

Protocols Used:

Restriction digest of pUniprom with BamHI/PstI for subsequent insertion of ChrB and ChrB-opt.

p>Results: Figure 17 Gel image

Next Steps:Insertion of ChrB and ChrB-opt respectively via BamHI/PstI.

Day 3 - 22/07

Aim of experiment:Ligation of pChr into pSB1C3 via Eco/Spe link.

Protocols Used:

Ligation of pChr(08/07/15) into pSB1C3(13/07/15)

Results: N/A

Aim of experiment:Transformation

Protocols Used:

Transformation of pSB1C3-pChr into JM110.

Results: Figure 18 Plates after transformation of pChr into pSB1C3.

Next Steps:Sequence confirmation of pChr in pSB1C3.

Day 4 - 23/07

Aim of experiment: To prepare pSB1C3-pChr for sequence confirmation

Protocols Used:

Overnight culture of pSB1C3-pChr.

Patch-plating of the same colonies of pSB1C3-pChr.

Results: N/A

Next Steps: Purification of the recombinant vector for sequence confirmation.

Day 5 - 24/07

Aim of experiment:Purification of pSB1C3-pChr for sequence confirmation.

Protocols Used:

Miniprep of pSB1C3-pChr.

Results: Insert table here

Aim of experiment:Sequence confirmation of pSB1C3-pChr.

Protocols Used:

Sequencing of samples 1 and 2 of pSB1C3-pChr.

Results: Figure 19 Alignment of sequenced pSB1C3-pChr vectors with pChr sequence.

Next Steps: Digest of pSB1C3-pChr with SpeI/PstI and ligation of GFPmut2.

Day 6

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 7

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Week Beginning 22/06

Summary

This week was all about getting started with the newly arrived biobrick BBa_K1058008

Day 1

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 2

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 3

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 4

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 5

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 6

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 7

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Week Beginning 22/06

Summary

This week was all about getting started with the newly arrived biobrick BBa_K1058008

Day 1

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 2

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 3

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 4

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 5

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 6

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 7

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Week Beginning 22/06

Summary

This week was all about getting started with the newly arrived biobrick BBa_K1058008

Day 1

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 2

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 3

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 4

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 5

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 6

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 7

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Week Beginning 22/06

Summary

This week was all about getting started with the newly arrived biobrick BBa_K1058008

Day 1

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 2

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 3

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 4

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 5

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 6

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 7

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Week Beginning 22/06

Summary

This week was all about getting started with the newly arrived biobrick BBa_K1058008

Day 1

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 2

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 3

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 4

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 5

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 6

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 7

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.