Team:Warsaw/Calendar-Main/8 July 2009
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<h3>Gradient PCR Pho<h3> | <h3>Gradient PCR Pho<h3> | ||
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<br /> | <br /> | ||
<p>Methods:</p> | <p>Methods:</p> | ||
- | <ul><li><p>PCR mixture's composition:</p> | + | <ul><li><p>PCR mixture's composition:</p> |
+ | <pre>1μl pfu buffer (Fermentas) | ||
+ | 1μl MgSO4 (Fermentas) | ||
+ | 0,5μl primers | ||
+ | 0,5μl dNTPs (10 mM) | ||
+ | 0,25μl pfu turbo polymerase | ||
+ | 0,5μl template DNA from Listeria??? (Salmonella) | ||
+ | optionally: 0,75μl DMSO</pre> | ||
+ | The solution was topped up with H2O to 10μl. | ||
</li> | </li> | ||
</ul> | </ul> | ||
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<br /> | <br /> | ||
<p>Results:</p> | <p>Results:</p> | ||
- | <img src=""/> | + | <img src="https://static.igem.org/mediawiki/2009/9/9b/2009_07_09_pho.JPG"/> |
<var> | <var> | ||
<ul> | <ul> | ||
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<li>control -</li> | <li>control -</li> | ||
<li>2-6 - samples (annealing temperature increases to from the left to the right)</li> | <li>2-6 - samples (annealing temperature increases to from the left to the right)</li> | ||
- | <li> | + | <li>M - GeneRuler DNA Ladder Mix #SM0333 (Fermentas) |
+ | <li>8-12 - samples with DMSO (annealing temperature increases to from the left to the right) | ||
<li>M - GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li> | <li>M - GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li> | ||
+ | </ol> | ||
<br/> | <br/> | ||
<p>Notes:</p> | <p>Notes:</p> | ||
- | + | <ul> | |
- | <li></li> | + | <li>many unspecific products were obtained <b>(product of the correct lenght marked with an arrow)</b></li> |
</ul> | </ul> | ||
</var> | </var> | ||
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</p> | </p> | ||
- | + | <h3>Quality check (cro PCR product and pKS isolate)</h3> | |
<p><br /> | <p><br /> | ||
<br /> | <br /> | ||
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<br /> | <br /> | ||
</html> | </html> | ||
+ | <html> | ||
+ | <h3>Cloning of p53 coding sequence</h3> | ||
+ | <h4>Marcin</h4> | ||
- | + | <p><strong>Comment:</strong></p> | |
- | + | <p>There were some difficulties with ligation p53 coding sequence into pKS plasmid so I decided to take another sample containing isolate amplified p53 (via PCR reaction) and perform digest of the sample using XbaI</p> | |
- | + | <br/> | |
- | + | <p>Tasks:</p> | |
- | + | <br/> | |
- | There were some difficulties with ligation p53 coding sequence into pKS plasmid so I decided to take another sample containing isolate amplified p53 (via PCR reaction) and perform digest of the sample using XbaI | + | <ul> |
- | + | <li>Restriction digest of p53 coding sequence obtained from PCR reaction</li> | |
- | Tasks: | + | </ul> |
- | + | <br/> | |
- | + | <p>Methods:</p> | |
- | + | <br/> | |
- | Methods: | + | <ul> |
- | + | <li>Reaction mixture composition</li> | |
- | + | </ul> | |
- | 22.5 | + | <p> |
- | + | <pre>22.5 μl PCR product (DNA concentration about 6.5 ng/μll) | |
- | Digest | + | 2.5 μl Tango Buffer (Fermentas) |
- | <pre> 3h 37 °C | + | 0.5 μl XbaI (Fermentas)</pre> |
- | 15 min 80 °C | + | </p> |
- | ~4 °C | + | <ul> |
+ | <li>Digest program:</li> | ||
+ | </ul> | ||
+ | <p>Digest</p> | ||
+ | <pre> 3h 37 °C<br/> | ||
+ | 15 min 80 °C<br/> | ||
+ | ~4 °C<br/> | ||
</pre> | </pre> | ||
+ | <br/> | ||
+ | <ul> | ||
+ | <li>Quantification of the amount of DNA after digest on the agarose gel</li> | ||
+ | </ul> | ||
+ | <br/> | ||
+ | <p>Methods:</p> | ||
+ | <ul> | ||
+ | <li>Electrophoresis of the digested DNA sample:</li> | ||
+ | </ul> | ||
+ | <p>Conditions:</p> | ||
+ | <p>agarose concentration - 1%</p> | ||
+ | <p>voltage - 70V</p> | ||
+ | <p>time - about 30 minutes</p> | ||
+ | <br/> | ||
+ | <p>After electrophoresis gel was irradiated with UV light and photographed:</p> | ||
+ | <img src="https://static.igem.org/mediawiki/2009/4/4b/P53_08_07_09.jpg" align="center" widht="80%" height="80%"> | ||
+ | <br/> | ||
+ | There is no significant amount of DNA in the samples | ||
- | + | <p><strong>Comment:</strong></p> | |
- | + | ||
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+ | <p>The sample must have degraded after gel-out procedure. Most probably due to acidic condition in the solution DNA hydrolysed. It is obligatory to do another PCR reaction using the old plasmid sample (I hope the DNA is not degraded!).</p> | ||
+ | </html> | ||
<!-- TU EDYTOWALA ANIA prosze nie szatkowac znacznikami! --> | <!-- TU EDYTOWALA ANIA prosze nie szatkowac znacznikami! --> | ||
<html> | <html> | ||
- | <h3>Miecznikowa team Aim: debug RFP-terminator ligation that did not work<h3> | + | <h3>Miecznikowa team Aim: debug RFP-terminator ligation that did not work</h3> |
<h4>Jarek/Franek/Ania</h4> | <h4>Jarek/Franek/Ania</h4> | ||
<p>Task1:</p> | <p>Task1:</p> | ||
Line 204: | Line 219: | ||
<ul> | <ul> | ||
<li>Rfptra and RBSdigested samples were extracted from the gel using gel-out kit by A&ABiotechnology. Presented data shows that after gel extraction there is very little DNA in the sample. | <li>Rfptra and RBSdigested samples were extracted from the gel using gel-out kit by A&ABiotechnology. Presented data shows that after gel extraction there is very little DNA in the sample. | ||
- | < | + | <br/><div><strong>Probably there is a ****problem with gel extraction procedure**** - the Gel-out set by A&A Biotechnology might be defective.</strong></div><br/> |
</li> | </li> | ||
</ul> | </ul> | ||
- | < | + | |
+ | <strong><p>Miecznikowa team - division of labour;)</p></strong> | ||
<p>Today the intermediate bricks were designed and we divided the tasks as follows:</p> | <p>Today the intermediate bricks were designed and we divided the tasks as follows:</p> | ||
<ul> | <ul> | ||
<li> | <li> | ||
- | Franek: BBa_J5528, digestion with (EcoRV, PstI) and insertion into pKS2 (Bluescript) cut with SmaI i PstI (look for white colonies on the X-gal, IPTG, Amp plates) | + | Franek: <a href="http://partsregistry.org/Part:BBa_J5528"><span style="color: black">BBa_J5528</a></span>, digestion with (EcoRV, PstI) and insertion into pKS2 (Bluescript) cut with SmaI i PstI (look for white colonies on the X-gal, IPTG, Amp plates) |
</li> | </li> | ||
<li> | <li> | ||
- | Jarek: construct BBa_K177011 | + | Jarek: construct <a href="http://partsregistry.org/Part:BBa_K177011"><span style="color: black">BBa_K177011</a></span> |
</li> | </li> | ||
<li> | <li> | ||
- | Michał: construct BBa_K177012 | + | Michał: construct <a href="http://partsregistry.org/Part:BBa_K177012"><span style="color: black">BBa_K177012</a></span> |
</li> | </li> | ||
<li> | <li> | ||
- | Marek: construct | + | Marek: construct <a href="http://partsregistry.org/Part:BBa_K177013"><span style="color: black">BBa_K177013</a></span> |
</li> | </li> | ||
<li> | <li> | ||
- | Ania: construct BBa_K177014 | + | Ania: construct <a href="http://partsregistry.org/Part:BBa_K177014"><span style="color: black">BBa_K177014</a></span> |
</li> | </li> | ||
<li> | <li> | ||
- | Monika: transform competent cells with | + | Monika: transform competent cells with <a href="http://partsregistry.org/Part:BBa_K1763004"><span style="color: black">BBa_K1763004</a></span>, <a href="http://partsregistry.org/Part:BBa_S03473"><span style="color: black">BBa_S03473</a></span>, <a href="http://partsregistry.org/Part:BBa_E0840"><span style="color: black">BBa_E0840</a></span>, <a href="http://partsregistry.org/Part:BBa_J07037"><span style="color: black">BBa_J07037</a></span> out of the distribution. |
</li> | </li> | ||
</ul> | </ul> | ||
<p>potential problems: get inv/llo/phoP/phoQ</p> | <p>potential problems: get inv/llo/phoP/phoQ</p> | ||
- | < | + | </strong></div> |
</html> | </html> | ||
<!-- TU EDYTUJE FRANEK, NIE RUSZ! --> | <!-- TU EDYTUJE FRANEK, NIE RUSZ! --> | ||
- | + | ||
- | <h3> | + | <h3>Creating devices to test promoters in <em>E. coli</em> strains (devices [http://partsregistry.org/Part:BBa_K177024 <span style="color: black;">BBa_K177024</span>] and [http://partsregistry.org/Part:BBa_K177025 <span style="color: black;">BBa_K177025</span>])</h3> |
<h4>Franek</h4> | <h4>Franek</h4> | ||
+ | <html> | ||
<br> | <br> | ||
<p>Task:</p> | <p>Task:</p> | ||
<ul> | <ul> | ||
- | <li>transform competent cells with <a href="http://partsregistry.org/Part:BBa_I0500">BBa_I0500</a><!-- to nieporozumienie miedzy mna a Michalem i izolowalem bezsensowny brick...! --></li> | + | <li>transform competent cells with <a href="http://partsregistry.org/Part:BBa_I0500"><font color="black">BBa_I0500</font></a><!-- to nieporozumienie miedzy mna a Michalem i izolowalem bezsensowny brick...! --></li> |
</ul> | </ul> | ||
<br> | <br> | ||
<p>Methods:</p> | <p>Methods:</p> | ||
<ul> | <ul> | ||
- | <li>Resuspension of DNA from plate 1, 14N (<a href="http://partsregistry.org/Part:BBa_I0500">BBa_I0500</a>) with 15µl of H2O</li> | + | <li>Resuspension of DNA from plate 1, 14N (<a href="http://partsregistry.org/Part:BBa_I0500"><font color="black">BBa_I0500</font></a>) with 15µl of H2O</li> |
- | <li>Transformation of chemocompetent cells with 4µl of <a href="http://partsregistry.org/Part:BBa_I0500">BBa_I0500</a> DNA solution</li> | + | <li>Transformation of chemocompetent cells with 4µl of <a href="http://partsregistry.org/Part:BBa_I0500"><font color="black">BBa_I0500</font></a> DNA solution</li> |
<li>Plating bacterias on LB medium supplemented with kanamycin</li> | <li>Plating bacterias on LB medium supplemented with kanamycin</li> | ||
</ul> | </ul> |
Latest revision as of 21:03, 19 September 2009
Gradient PCR Pho
Kama
Kama
Tasks:
- Amplification of phoP/phoQ
Methods:
PCR mixture's composition:
1μl pfu buffer (Fermentas) 1μl MgSO4 (Fermentas) 0,5μl primers 0,5μl dNTPs (10 mM) 0,25μl pfu turbo polymerase 0,5μl template DNA from Listeria??? (Salmonella) optionally: 0,75μl DMSO
The solution was topped up with H2O to 10μl.
- PCR programs:
pho
4min 95°C
(30s 95°C, 1min 45-55°C, 4min 72°C)x3
(30s 95°C, 1min 55-60°C, 4min 72°C)x28
10min 72°C
~ 7°C
- Electrophoretic separation on 1% agarose gel
Results:
- Gel (from left)
- control -
- 2-6 - samples (annealing temperature increases to from the left to the right)
- M - GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
- 8-12 - samples with DMSO (annealing temperature increases to from the left to the right)
- M - GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
Notes:
- many unspecific products were obtained (product of the correct lenght marked with an arrow)
Quality check (cro PCR product and pKS isolate)
Kuba
- pKS-CRO cut with XbaI and EcoRI
- Gel (from the left)
- GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
- uncut plasmid (isolate no.1)
- cut plasmid (isolate no.1)
- uncut plasmid (isolate no.2)
- cut plasmid (isolate no.2)
- uncut plasmid (isolate no.3)
- cut plasmid (isolate no.3)
Note: isolate no.1 contains a correctly inserted PCR product
Cloning of p53 coding sequence
Marcin
Comment:
There were some difficulties with ligation p53 coding sequence into pKS plasmid so I decided to take another sample containing isolate amplified p53 (via PCR reaction) and perform digest of the sample using XbaI
Tasks:
- Restriction digest of p53 coding sequence obtained from PCR reaction
Methods:
- Reaction mixture composition
22.5 μl PCR product (DNA concentration about 6.5 ng/μll) 2.5 μl Tango Buffer (Fermentas) 0.5 μl XbaI (Fermentas)
- Digest program:
Digest
3h 37 °C
15 min 80 °C
~4 °C
- Quantification of the amount of DNA after digest on the agarose gel
Methods:
- Electrophoresis of the digested DNA sample:
Conditions:
agarose concentration - 1%
voltage - 70V
time - about 30 minutes
After electrophoresis gel was irradiated with UV light and photographed:
There is no significant amount of DNA in the samples
Comment:
The sample must have degraded after gel-out procedure. Most probably due to acidic condition in the solution DNA hydrolysed. It is obligatory to do another PCR reaction using the old plasmid sample (I hope the DNA is not degraded!).
Miecznikowa team Aim: debug RFP-terminator ligation that did not work
Jarek/Franek/Ania
Task1:
- Alkaline lysis of the plasmid containing Terminator
Methods:
PlasmidMini set by A&A Biotechnology was used. 2 test tubes with 5ml LB and 5 ul Ampicyline were first inoculated with the plasmid containing colonies. The cultures were incubated over night at 37C.Alkaline lysis was performed on both cultures. The pellet from 5 ml of bacteria was used.
Results:
- DNA extraction quality controll.DNA Concentration was measured using Spectrophotometer NanoDrop ND-1000
DNA sample | DNA concentration in ng |
---|---|
Terminator sample 1 (Ter1) | 73.9 ng/ul |
Terminator sample 1 (Ter2) | 85.46 ng/ul |
Task2:
- We decided to measure the concentration of DNA in our samples for future use e.g. efficient ligation mix. NanoDrop ND-1000 was used.
Results:
DNA sample | DNA concentration in ng |
---|---|
Rfp3 1st measurement | 17.49 ng/ul |
Rfp3 2nd | 24.18 ng/ul |
Rfp3 3rd | 24.50 ng/ul |
Rfp4 1st measurement | 28.85 ng/ul |
Rfptra (digested plasmid with RFP) | 0.4 ng/ul |
Rfptra 2nd measurement | 0.8 ng/ul |
RBS digested (digested RBS) | 1.44 ng/ul |
RBS digested 2nd measurement | 0.72 ng/ul |
Notes - IMPORTANT:
- Rfptra and RBSdigested samples were extracted from the gel using gel-out kit by A&ABiotechnology. Presented data shows that after gel extraction there is very little DNA in the sample.
Probably there is a ****problem with gel extraction procedure**** - the Gel-out set by A&A Biotechnology might be defective.
Miecznikowa team - division of labour;)
Today the intermediate bricks were designed and we divided the tasks as follows:
- Franek: BBa_J5528, digestion with (EcoRV, PstI) and insertion into pKS2 (Bluescript) cut with SmaI i PstI (look for white colonies on the X-gal, IPTG, Amp plates)
- Jarek: construct BBa_K177011
- Michał: construct BBa_K177012
- Marek: construct BBa_K177013
- Ania: construct BBa_K177014
- Monika: transform competent cells with BBa_K1763004, BBa_S03473, BBa_E0840, BBa_J07037 out of the distribution.
potential problems: get inv/llo/phoP/phoQ
Creating devices to test promoters in E. coli strains (devices [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] and [http://partsregistry.org/Part:BBa_K177025 BBa_K177025])
Franek
Task:
- transform competent cells with BBa_I0500
Methods:
- Resuspension of DNA from plate 1, 14N (BBa_I0500) with 15µl of H2O
- Transformation of chemocompetent cells with 4µl of BBa_I0500 DNA solution
- Plating bacterias on LB medium supplemented with kanamycin
Results:
- Will be determined tomorrow
Jarek
Task:
- Preparation of bacterial cultures containing parts R0010, B0032 and C0051 in LB with ampicilin
Methods:
- Concentration of ampicilin used for cultures was 100 ug/ml
Results:
- The growth of the cultures will be observed on the next day
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