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"> | ||
− | Part: <strong> | + | Part: <strong>P30</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> | + | Performed by: <strong>ŠT</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>cI repressor promoter (BBa_R1051) (P4) + screen plasmid intermediate (GFP gene) (BBa_I13504) (P5).</p> |
</div> | </div> | ||
</div> | </div> | ||
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<tr> | <tr> | ||
<td>06 30</td> | <td>06 30</td> | ||
− | <td> | + | <td>P14</td> |
− | <td> | + | <td>P4 transformation into DH5α.</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
Line 404: | Line 404: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>07 | + | <td>07 02</td> |
− | <td> | + | <td>P4</td> |
<td> | <td> | ||
− | + | Plasmid extraction. Concentrations:<br /> | |
− | + | P4 - 238,3 ng/μL and 224,3 ng/μL.<br /> | |
− | + | P4 restriction with EcoRI and XBal.<br /> | |
− | Extraction from gel<br/> | + | Extraction from gel.<br /> |
− | + | L4 is produced (con. 16,2 ng/μL). | |
− | + | ||
</td> | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>07 07</td> | ||
+ | <td>P5</td> | ||
+ | <td> | ||
+ | P5 transformation into DH5α. <br /> | ||
+ | Bacteria inoculation. | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>07 08</td> | ||
+ | <td></td> | ||
+ | <td>P5 plasmid extraction (P5.1, P5.2, P5.3, P5.4, P5.5 are produced).</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>07 29</td> | ||
+ | <td>P5.1 and <br />P5.3</td> | ||
+ | <td>Restriction with EcoRI and SpeI.<br /> | ||
+ | Extraction from gel.<br /> | ||
+ | L5.1A, L5.1B, L5.1C and <br /> | ||
+ | L5.3A, L5.3B, L5.3C are produced.<br /> | ||
+ | Conc.: L5.1A – 0,6 ng/μL, L5.1B – 0,6 ng/μL, L5.1C – 2,4 ng/μL, <br /> | ||
+ | L5.3A – 0,7 ng/μL, L5.3B - 3 ng/μL, L5.3C – 5,1 ng/μL.<br /> | ||
+ | Ligation of L4 and L5.1, L4 and L5.3.<br /> | ||
+ | P30 is produced.</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>08 01</td> | ||
+ | <td>P30</td> | ||
+ | <td>Sequencing.</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 09 – fragments were not the right ones.<br /> 07 29 – the right fragments are produced.</p> |
</div> | </div> | ||
</div> | </div> | ||
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<div class="row"> | <div class="row"> | ||
<div class="col-md-6"> | <div class="col-md-6"> | ||
− | Part: <strong> | + | Part: <strong>P35, P36, P37</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> |
+ | P35 - pLux/cI+SRBS(Strong RBS)+Cas3 (L26+L15.1)<br /> | ||
+ | P36 - pLux/cI+MRBS(Medium RBS)+Cas3 (L27+L15.1)<br /> | ||
+ | P37 - pLux/cI+WRBS(Weak RBS)+Cas3 (L28+L15.1)<br /> | ||
+ | P26 – L7+L26 (pLux/cI+SRBS)<br /> | ||
+ | P27 - pLux/cI+MRBS(Medium RBS)<br /> | ||
+ | P28 - pLux/cI+WRBS(Weak RBS)</p> | ||
</div> | </div> | ||
</div> | </div> | ||
Line 471: | Line 506: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
− | <td> | + | <td>07 28</td> |
− | <td> | + | <td>P15</td> |
− | <td> | + | <td>Mutated Cas3 with Preffix and Suffix restriction with EcoRI and XbaI.<br /> |
+ | L15 is produced.</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>07 | + | <td>07 28</td> |
− | <td></td> | + | <td> |
− | <td> | + | P26<br /> |
+ | P27<br /> | ||
+ | P28</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> | ||
− | + | L15<br /> | |
− | + | L26<br /> | |
− | + | L27<br /> | |
− | + | L28</td> | |
− | + | <td>Ligation of: L15 and L26, L15 and L27, L15 and L28, <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 /> | ||
+ | P35, P36 and P37are produced<br /> | ||
+ | P35, P36, P37 restriction with NspI.</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>08 10</td> | ||
+ | <td></td> | ||
+ | <td>P35, P36, P37 sequencing.</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 22:19, 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.