Difference between revisions of "Team:Vilnius-Lithuania/Labjournal"
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<p style="height: auto; margin: auto;">07 31 – all of the colonies are successful.<br /> | <p style="height: auto; margin: auto;">07 31 – all of the colonies are successful.<br /> | ||
08 10 – all biobricks are sequenced succesfully.</p> | 08 10 – all biobricks are sequenced succesfully.</p> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | <!-- /MethodBox --> | ||
+ | |||
+ | <!-- MethodBox --> | ||
+ | <div class="panel panel-default" style="position: relative"> | ||
+ | <div class="panel-heading"> | ||
+ | <a class="accordion-toggle collapsed" data-toggle="collapse" data-parent="#accordion" href="#2"> | ||
+ | <div class="panel-title"> | ||
+ | <h3 style="padding: 0px; margin: 0px;">Parts construction</h3> | ||
+ | <div class="row"> | ||
+ | <div class="col-md-6"> | ||
+ | Part: <strong>Cd1</strong> (BBa_) | ||
+ | </div> | ||
+ | <div class="col-md-6"> | ||
+ | Written by: <strong>IR</strong><br /> | ||
+ | Performed by: <strong>ŠT</strong>, <strong>IR</strong>, <strong>MB</strong>, <strong>IS</strong>, <strong>IO</strong>. | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </a> | ||
+ | </div> | ||
+ | <div id="2" class="panel-collapse collapse"> | ||
+ | <div class="panel-body"> | ||
+ | <div class="row"> | ||
+ | <div class="col-md-12"> | ||
+ | <h3><strong>About</strong></h3> | ||
+ | <p>Mutated Cascade protein.</p> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="row"> | ||
+ | <div class="col-md-12"> | ||
+ | <table class="table"> | ||
+ | <thead> | ||
+ | <tr> | ||
+ | <th>Date</th> | ||
+ | <th>Materials</th> | ||
+ | <th>Experiment</th> | ||
+ | </tr> | ||
+ | </thead> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>06 30</td> | ||
+ | <td>Cd</td> | ||
+ | <td>Cd transformation into DH5α.</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>07 01</td> | ||
+ | <td></td> | ||
+ | <td>Transformants are inoculated (4 minipreps each).</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>07 02</td> | ||
+ | <td></td> | ||
+ | <td>Plasmid extraction. Conc.: 329 ng/μL and 294 ng/μL. | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>07 14</td> | ||
+ | <td>Cd</td> | ||
+ | <td>Cd mutagenesis (with ST13/14 primers).</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>07 16 - 17</td> | ||
+ | <td></td> | ||
+ | <td>Mutated plasmids extraction. Restriction with EcoRI.</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>07 19</td> | ||
+ | <td>Mutated Cd</td> | ||
+ | <td>Restriction repeat.</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>07 21</td> | ||
+ | <td>Mutated Cd</td> | ||
+ | <td>Repeated mutagenesis (with ST13/14).</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>07 27</td> | ||
+ | <td>Mutated Cd</td> | ||
+ | <td>Mutated plasmids extraction. Mutated Cd restriction.</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>08 10</td> | ||
+ | <td>Mutated Cd <br /> L7</td> | ||
+ | <td> | ||
+ | PCR with mutated Cd with ST26/27.<br /> | ||
+ | PCR product purification.<br /> | ||
+ | Conc.: 63 ng/μL.<br /> | ||
+ | Restriction with EcoRI and SpeI.<br /> | ||
+ | Mutated Cd ligation with L7.<br /> | ||
+ | Cd1 is produced.</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td></td> | ||
+ | <td>Cd1</td> | ||
+ | <td>Sequencing.</td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="row"> | ||
+ | <div class="col-md-12"> | ||
+ | <h3><strong>Conclusions and Results:</strong></h3> | ||
+ | <p style="height: auto; margin: auto;">07 16-17 – Mutagenesis was unsuccessful.<br /> | ||
+ | 07 27 – Mutagenesis conformed as a successful.</p> | ||
</div> | </div> | ||
</div> | </div> |
Revision as of 22:35, 7 September 2015
About
pLac+cI.
P10 and P11 plasmids are used. pLac (P11) and repressor cI (P10) ligation.
Date | Materials | Experiment |
---|---|---|
06 30 | P10 P11 |
P10 and P11 transformation into DH5α. |
07 01 | Bacteria inoculation (4 minipreps). | |
07 09 | P10 P11 |
Plasmids’ restriction (3 ug each). P10 with EcoRI and XBal (+FastAp). P11 with EcoRI and SpeI. Extraction from gel L10 and L11 are produced. Conc.: L11 – 2,4 ng/uL. |
Conclusions and Results:
07 14 – all of the colonies are successful, except third colony.
About
Cas3 with mutated restriction sites
L7+L15. Concentration - 261 ng/μL
Date | Materials | Experiment |
---|---|---|
06 30 | P13 P7 |
P13 and P7 transformation into DH5α. |
07 01 | Transformants are inoculated (4 minipreps each). | |
07 02 | Plasmid extraction. Concentrations: P7 – 174,1 ng/μL and 166 ng/μL. P13 - 216 ng/μL and 131, 5 ng/μL. |
|
07 13 | P13 | Cas3 mutagenesis (with ST1/2, ST3/4, ST5/6 primers). |
07 16 | Mutated P13 | Cas3 plasmids extraction. |
07 21-23 | Mutated P13 | Restriction with EcoRI, XbaI and PstI. |
07 27 | Mutated P13 | Cas3 mutagenesis. |
07 29 | P7 | Restriction with EcoRI and SpeI. L7 is produced. |
07 31 | P13 | |
08 02 | Mutated P13 | Mutated Cas3 restriction with EcoRI, XbaI and PstI. |
08 05 | Mutated P13 | Cas3 mutagenesis. |
08 07 | Mutated P13 | Mutated Cas3 restriction with PstI. Mutagenesis was successful. L15 is produced. |
08 11 | L7 L15 |
L7 and L15 ligation with pSB1C3 (EcoRI+XBal+FastAp). P15.1 is produced. |
P15.1 | Sequencing. |
Conclusions and Results:
07 21 – Mutagenesis was unsuccessful.
08 02 – successfully mutagenized EcoRI and XbaI sites.
08 07 – successful mutagenesis confirmed (XbaI and EcoRI sites are mutated).
08 07 – Successful mutagenesis (PstI, XbaI and EcoRI sites are mutated).
About
P26 – L7+L26 pLux/cI+SRBS
P27 – L7+L27 pLux/cI+MRBS
P28 – L7+L28 pLux/cI+WRBS
P29 – L7+L29 pLux/cI
Date | Materials | Experiment |
---|---|---|
07 27 | ST9 ST10 ST11 ST12 ST15 ST16 ST17 ST18 ST19 ST20 |
pLux/cI contruction from oligos – PCR reaction with ST9 and ST10/11/12 primers, then with ST15 and ST16/17/18. Parallel PCR with ST9 and ST19 and then with ST15 and ST20. PCR products purification. |
07 28 | PCR products from oligos restriction with EcoRI and SpeI. L26, L27, L28 and L29 are produced. |
|
07 28 | L7 L26 L27 L28 L29 |
Ligation of: L7 and L26, L7 and L27, L7 and L28, L7 and L29 and their transformation into DH5α. |
07 29 - - 08 05 |
L7+L26 colony PCR with ST15 and VR. L7+L27 colony PCR with ST15 and VR. L7+L28 colony PCR with ST15 and VR. 1st and 2nd colonies are inoculated. P26, P27, 28 and P29 are produced P26, P27, 28 and P29 restriction with NspI. |
|
08 10 | P26, P27, 28 and P29 sequencing with VF2. |
Conclusions and Results:
07 31 – all of the colonies are successful.
08 10 – all biobricks are sequenced succesfully.
About
cI repressor promoter (BBa_R1051) (P4) + screen plasmid intermediate (GFP gene) (BBa_I13504) (P5).
Date | Materials | Experiment |
---|---|---|
06 30 | P14 | P4 transformation into DH5α. |
07 01 | Bacteria inoculation (4 minipreps). | |
07 02 | P4 |
Plasmid extraction. Concentrations: P4 - 238,3 ng/μL and 224,3 ng/μL. P4 restriction with EcoRI and XBal. Extraction from gel. L4 is produced (con. 16,2 ng/μL). |
07 07 | P5 |
P5 transformation into DH5α. Bacteria inoculation. |
07 08 | P5 plasmid extraction (P5.1, P5.2, P5.3, P5.4, P5.5 are produced). | |
07 29 | P5.1 and P5.3 |
Restriction with EcoRI and SpeI. Extraction from gel. L5.1A, L5.1B, L5.1C and L5.3A, L5.3B, L5.3C are produced. Conc.: L5.1A – 0,6 ng/μL, L5.1B – 0,6 ng/μL, L5.1C – 2,4 ng/μL, L5.3A – 0,7 ng/μL, L5.3B - 3 ng/μL, L5.3C – 5,1 ng/μL. Ligation of L4 and L5.1, L4 and L5.3. P30 is produced. |
08 01 | P30 | Sequencing. |
Conclusions and Results:
07 09 – fragments were not the right ones.
07 29 – the right fragments are produced.
About
P35 - pLux/cI+SRBS(Strong RBS)+Cas3 (L26+L15.1)
P36 - pLux/cI+MRBS(Medium RBS)+Cas3 (L27+L15.1)
P37 - pLux/cI+WRBS(Weak RBS)+Cas3 (L28+L15.1)
P26 – L7+L26 (pLux/cI+SRBS)
P27 - pLux/cI+MRBS(Medium RBS)
P28 - pLux/cI+WRBS(Weak RBS)
Date | Materials | Experiment |
---|---|---|
07 28 | P15 | Mutated Cas3 with Preffix and Suffix restriction with EcoRI and XbaI. L15 is produced. |
07 28 |
P26 P27 P28 |
PCR products from oligos restriction with EcoRI and SpeI. L26, L27, L28 and L29 are produced. |
07 28 |
L15 L26 L27 L28 |
Ligation of: L15 and L26, L15 and L27, L15 and L28, and their transformation into DH5α. |
07 29 - - 08 05 |
L7+L26 colony PCR with ST15 and VR. L7+L27 colony PCR with ST15 and VR L7+L28 colony PCR with ST15 and VR 1st and 2nd colonies are inoculated. P35, P36 and P37are produced P35, P36, P37 restriction with NspI. |
|
08 10 | P35, P36, P37 sequencing. |
Conclusions and Results:
07 31 – all of the colonies are successful.
08 10 – all biobricks are sequenced succesfully.
About
Mutated Cascade protein.
Date | Materials | Experiment |
---|---|---|
06 30 | Cd | Cd transformation into DH5α. |
07 01 | Transformants are inoculated (4 minipreps each). | |
07 02 | Plasmid extraction. Conc.: 329 ng/μL and 294 ng/μL. | |
07 14 | Cd | Cd mutagenesis (with ST13/14 primers). |
07 16 - 17 | Mutated plasmids extraction. Restriction with EcoRI. | |
07 19 | Mutated Cd | Restriction repeat. |
07 21 | Mutated Cd | Repeated mutagenesis (with ST13/14). |
07 27 | Mutated Cd | Mutated plasmids extraction. Mutated Cd restriction. |
08 10 | Mutated Cd L7 |
PCR with mutated Cd with ST26/27. PCR product purification. Conc.: 63 ng/μL. Restriction with EcoRI and SpeI. Mutated Cd ligation with L7. Cd1 is produced. |
Cd1 | Sequencing. |
Conclusions and Results:
07 16-17 – Mutagenesis was unsuccessful.
07 27 – Mutagenesis conformed as a successful.