Difference between revisions of "Team:Vilnius-Lithuania/Labjournal"
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<div class="row"> | <div class="row"> | ||
<div class="col-md-6"> | <div class="col-md-6"> | ||
− | + | Parts: <strong>P26, P27, P28, P29</strong> (BBa_) | |
</div> | </div> | ||
<div class="col-md-6"> | <div class="col-md-6"> | ||
Written by: <strong>IR</strong><br /> | Written by: <strong>IR</strong><br /> | ||
− | Performed by: <strong>ŠT</strong>, <strong>IR</strong>, <strong> | + | Performed by: <strong>ŠT</strong>, <strong>IR</strong>, <strong>BB</strong>, <strong>IS</strong>. |
</div> | </div> | ||
</div> | </div> | ||
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<div class="col-md-12"> | <div class="col-md-12"> | ||
<h3><strong>About</strong></h3> | <h3><strong>About</strong></h3> | ||
− | <p> | + | <p>P26 – L7+L26 pLux/cI+SRBS<br /> |
+ | P27 – L7+L27 pLux/cI+MRBS <br /> | ||
+ | P28 – L7+L28 pLux/cI+WRBS <br /> | ||
+ | P29 – L7+L29 pLux/cI</p> | ||
</div> | </div> | ||
</div> | </div> | ||
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<tbody> | <tbody> | ||
<tr> | <tr> | ||
− | <td> | + | <td>07 27</td> |
− | <td> | + | <td>ST9<br /> |
− | <td> | + | ST10<br /> |
+ | ST11<br /> | ||
+ | ST12<br /> | ||
+ | ST15<br /> | ||
+ | ST16<br /> | ||
+ | ST17<br /> | ||
+ | ST18<br /> | ||
+ | ST19<br /> | ||
+ | ST20</td> | ||
+ | <td>pLux/cI contruction from oligos – PCR reaction with ST9 and ST10/11/12 primers, then with <br />ST15 and ST16/17/18.<br /> | ||
+ | Parallel PCR with ST9 and ST19 and then with ST15 and ST20.<br /> | ||
+ | PCR products purification.</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>07 | + | <td>07 28</td> |
<td></td> | <td></td> | ||
− | <td> | + | <td>PCR products from oligos restriction with EcoRI and SpeI.<br /> L26, L27, L28 and L29 are produced.</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>07 | + | <td>07 28</td> |
− | <td> | + | <td>L7<br /> |
− | <td> | + | L26<br /> |
− | + | L27<br /> | |
− | + | L28<br /> | |
− | + | L29</td> | |
− | + | <td>Ligation of:<br /> | |
− | + | L7 and L26, L7 and L27, L7 and L28, L7 and L29 <br /> and their transformation into DH5α.</td> | |
− | + | </tr> | |
− | </td> | + | <tr> |
+ | <td>07 29 - <br /> - 08 05</td> | ||
+ | <td></td> | ||
+ | <td>L7+L26 colony PCR with ST15 and VR.<br /> | ||
+ | L7+L27 colony PCR with ST15 and VR.<br /> | ||
+ | L7+L28 colony PCR with ST15 and VR.<br /> | ||
+ | 1st and 2nd colonies are inoculated.<br /> | ||
+ | P26, P27, 28 and P29 are produced<br /> | ||
+ | P26, P27, 28 and P29 restriction with NspI.</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>08 10</td> | ||
+ | <td></td> | ||
+ | <td>P26, P27, 28 and P29 sequencing with VF2.</td> | ||
</tr> | </tr> | ||
</tbody> | </tbody> | ||
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<div class="col-md-12"> | <div class="col-md-12"> | ||
<h3><strong>Conclusions and Results:</strong></h3> | <h3><strong>Conclusions and Results:</strong></h3> | ||
− | <p style="height: auto; margin: auto;">07 | + | <p style="height: auto; margin: auto;">07 31 – all of the colonies are successful.<br /> |
+ | 08 10 – all biobricks are sequenced succesfully.</p> | ||
</div> | </div> | ||
</div> | </div> |
Revision as of 21:59, 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
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
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.