Team:Warsaw/Calendar-Main/13 August 2009
From 2009.igem.org
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3.0 μl - primer BaxF (0.5 μM) | 3.0 μl - primer BaxF (0.5 μM) | ||
3.0 μl - primer BaxR2 (0.5 μM) | 3.0 μl - primer BaxR2 (0.5 μM) | ||
- | 1.0 μl - | + | 1.0 μl - template DNA |
32.0μl - mQ water | 32.0μl - mQ water | ||
2.5 μl - Yellow PfuPlus DNA polymerase (1U/μl) | 2.5 μl - Yellow PfuPlus DNA polymerase (1U/μl) | ||
Line 24: | Line 24: | ||
<li>Template DNA in 3 dilutions - 10<sup>1</sup> (pure plasmid DNA), 10<sup>-1</sup> and 10<sup>-2</sup>.</li> | <li>Template DNA in 3 dilutions - 10<sup>1</sup> (pure plasmid DNA), 10<sup>-1</sup> and 10<sup>-2</sup>.</li> | ||
<li>buffer, dNTP mix and polymerase from EURx.</li> | <li>buffer, dNTP mix and polymerase from EURx.</li> | ||
+ | <li>DNA template - human Bax with changed codons to be more efficient in yeast. </li> | ||
<br> | <br> | ||
<br> | <br> | ||
Line 48: | Line 49: | ||
<p>Results:</p> | <p>Results:</p> | ||
<li>Best reaction for 10<sup>1</sup> and 10<sup>-1</sup> (more specific), in 69.1°C and 69.8°C. | <li>Best reaction for 10<sup>1</sup> and 10<sup>-1</sup> (more specific), in 69.1°C and 69.8°C. | ||
+ | <br> | ||
+ | <br> | ||
+ | <center> | ||
<img src="https://static.igem.org/mediawiki/2009/e/e2/Baxpcr1.JPG"> | <img src="https://static.igem.org/mediawiki/2009/e/e2/Baxpcr1.JPG"> | ||
+ | </center> | ||
<br> | <br> | ||
<br> | <br> | ||
Line 54: | Line 59: | ||
</ul> | </ul> | ||
</html> | </html> | ||
+ | <html> | ||
+ | <h3><div style="text-align: center;">Cloning of the mgtc promoter into the pSB1A3 plasmid</div></h3> | ||
+ | <h4>Kamil</h4> | ||
+ | <br /> | ||
+ | <p>Tasks:</p> | ||
+ | <ul> | ||
+ | <li>Reamplification of the mgtc promoter</li> | ||
+ | </ul> | ||
+ | <br /> | ||
+ | <p>Methods:</p> | ||
+ | <ul> | ||
+ | <li>The PCR mix was prepared as follows: | ||
+ | <p><div align=center><pre>5μl buffer B (EURx)<br/> 2μl 5mM dNTPs (EURx)<br/> 5μl forward starter<br/> 5μl reverse starter<br/> 2μl OptiTaq polymerase (EURx)<br/> 2μl matrix <br/> 29 μl H<sub>2</sub>O </pre></li></ul> | ||
+ | <ul><li>PCR programme: | ||
+ | <p><div align=center><pre> 4min 95°C <br/> (30s 95°C, 35s 58°C, 40s 72°C)x28 <br/> 10min 72°C <br/> ~ 4°C<br/></pre></li></ul> | ||
+ | <ul> | ||
+ | <li>The results were visualised with gel electrophoresis on 1% agarose gel.</li> | ||
+ | <li>The appropriate bands were extracted using Gel-Out kit (A&A Biotechnology) according to the producers protocol. | ||
+ | </ul> | ||
+ | <br /> | ||
+ | <p>Results:</p> | ||
+ | <ul> | ||
+ | <li>Gel Electrophoresis:</li> | ||
+ | </ul> | ||
+ | <img src=https://static.igem.org/mediawiki/2009/3/30/2009.07.27.jpg /> | ||
+ | <p>From left: | ||
+ | <ol> | ||
+ | <li>GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li> | ||
+ | <li>50μl of PCR mix</li> | ||
+ | <li>50μl of PCR mix</li> | ||
+ | <li>Negative control</li> | ||
+ | </ol> | ||
+ | <br /> | ||
+ | <p>Conclusions:</p> | ||
+ | <ul> | ||
+ | <li>Reamplification was successful.</li> | ||
+ | </ul> | ||
+ | <br /> | ||
+ | </html> | ||
+ | |||
+ | <html> | ||
+ | |||
+ | <h3><div style="text-align: center;">Assembly of endosomal detection operon</div></h3> | ||
+ | <h4>Marcin</h4> | ||
+ | <br /> | ||
+ | <p>Task 1:</p> | ||
+ | <ul><li>Gel-out of samples which was cut out from the gel in <a href="https://2009.igem.org/Team:Warsaw/Calendar-Main/12_August_2009">12.08.09</a></li></ul> | ||
+ | <p>Procedure:<p/> | ||
+ | <ul> | ||
+ | <li>Fragments of agarose gel were carefully cut out and in the next step DNA was extracted from the gel using the A&A gel-out kit. Detailed procedure is described <a href="http://aabiot.com/products/dna_purification/dna_fragments/gel_out/protocol_gel_out.pdf">here</a></li></ul> | ||
+ | <p>Results:<p> | ||
+ | <center> | ||
+ | <img src="https://static.igem.org/mediawiki/2009/0/01/Gel_outs_13_08_09.png" width="40%" height="40%"></center> | ||
+ | <font face="Times New Roman" size="3"><p><div style="text-align: center;">Evaluation of gel-outs yield</div> | ||
+ | </p></font> | ||
+ | <p><b>Comment:</b></p> | ||
+ | <p>The concentration of DNA is sample containing <a href="http://partsregistry.org/Part:BBa_R0080"><span style="color: black">BBa_R0080</a></span> was surprisingly low (sample 1). Because of this I decided to repeat the digest and try to use another gel-out kit.</p><br/> | ||
+ | <p>Task 2:</p><ul><li>Restriction digest of <a href="http://partsregistry.org/Part:BBa_B0032"><span style="color: black">BBa_B0032</a></span></li></ul> | ||
+ | <p>Methods:<p/><ul> | ||
+ | <li>Digest of BBa_R0080 using SpeI and PstI</li> | ||
+ | <li> | ||
+ | Reaction mixture composition:</li><pre> | ||
+ | 20 μl purified plasmid DNA product | ||
+ | 0.5 μl SpeI (Fermentas) | ||
+ | 1 μl PstI (Fermentas) | ||
+ | 5 μl Buffer Tango (Fermentas) | ||
+ | 24 μl MQ water</pre> | ||
+ | </ul> | ||
+ | <br/> | ||
+ | <p>Task 3:</p><ul><li>Cloning the sequences: cro CDS and <a href="http://partsregistry.org/Part:BBa_E0022"><span style="color: black">BBa_E0022</a></span> with <a href="http://partsregistry.org/Part:BBa_B0032"><span style="color: black">BBa_B0032</a></span> vector</ul></li> | ||
+ | <br/> | ||
+ | <p>Methods:</p> | ||
+ | <ul> | ||
+ | <li> | ||
+ | Ligation mixtures composition:</li><pre> | ||
+ | 15 μl digested cro (or E0022) | ||
+ | 8 μl digested vector | ||
+ | 3 μl Tango buffer(Fermentas) | ||
+ | 3 μl dNTPs mixture (EurX, concentration 5 mM) | ||
+ | 1 μl ligase T4 (Fermentas)</pre> | ||
+ | <li>Duration of ligation was about 18 hours; reaction was conducted in 19 °c (approximately).</li> | ||
+ | <li>In the next step ligated samples were thermally inactivated via heating in 80 °c for 20 minutes</li> | ||
+ | </ul> | ||
+ | <br/> | ||
+ | <p>Task 4:</p><ul><li>Prepare the bacterial cultures for isolation of on <a href="http://partsregistry.org/Part:pSB1A3"><span style="color: black">pSB1A3</a></span> plasmid containing:</li> | ||
+ | <ul> | ||
+ | <li>p53</li> | ||
+ | <li><a href="http://partsregistry.org/Part:BBa_B0032"><span style="color: black">BBa_B0032</a></span>+<a href="http://partsregistry.org/Part:BBa_C0040"><span style="color: black">BBa_C0040</a></span></li> | ||
+ | <li><a href="http://partsregistry.org/Part:BBa_B0032"><span style="color: black">BBa_B0032</a></span>+<a href="http://partsregistry.org/Part:BBa_C0051"><span style="color: black">BBa_C0051</a></span></li> | ||
+ | <li><a href="http://partsregistry.org/Part:BBa_B0032"><span style="color: black">BBa_B0032</a></span>+<a href="http://partsregistry.org/Part:BBa_E0032"><span style="color: black">BBa_E0032</a></span></li></ul><br/> | ||
+ | </html> | ||
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{{WarNotebookEnd}} | {{WarNotebookEnd}} |
Latest revision as of 20:44, 13 September 2009
Amplyfing of Bax sequence
Justyna
Methods
- PCR reaction mix:
per 50μl:
5.0 μl - 10 x buffer (20mM MgSO4) 3.5 μl - dNTP mix (5mM) 3.0 μl - primer BaxF (0.5 μM) 3.0 μl - primer BaxR2 (0.5 μM) 1.0 μl - template DNA 32.0μl - mQ water 2.5 μl - Yellow PfuPlus DNA polymerase (1U/μl)
Thermal cycling conditions for PCR (~7-8 kb):
94°C, 1 min 0s 94°C, 0 min 15s 69°C-74°C, 0 min 30s 68°C, 1 min 0s cycles 1-10 94°C, 0 min 15s 69°C-74°C, 0 min 30s 68°C, 1 min 20s cycles 11-25 68°C, 7 min 0s 4°C, indefinite
Results:
Cloning of the mgtc promoter into the pSB1A3 plasmid
Kamil
Tasks:
- Reamplification of the mgtc promoter
Methods:
- The PCR mix was prepared as follows:
5μl buffer B (EURx)
2μl 5mM dNTPs (EURx)
5μl forward starter
5μl reverse starter
2μl OptiTaq polymerase (EURx)
2μl matrix
29 μl H2O- PCR programme:
4min 95°C
(30s 95°C, 35s 58°C, 40s 72°C)x28
10min 72°C
~ 4°C- The results were visualised with gel electrophoresis on 1% agarose gel.
- The appropriate bands were extracted using Gel-Out kit (A&A Biotechnology) according to the producers protocol.
Results:
- Gel Electrophoresis:
From left:
- GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
- 50μl of PCR mix
- 50μl of PCR mix
- Negative control
Conclusions:
- Reamplification was successful.
Assembly of endosomal detection operonMarcin
Task 1:
- Gel-out of samples which was cut out from the gel in 12.08.09
Procedure:
- Fragments of agarose gel were carefully cut out and in the next step DNA was extracted from the gel using the A&A gel-out kit. Detailed procedure is described here
Results:
Evaluation of gel-outs yieldComment:
The concentration of DNA is sample containing BBa_R0080 was surprisingly low (sample 1). Because of this I decided to repeat the digest and try to use another gel-out kit.
Task 2:
- Restriction digest of BBa_B0032
Methods:
- Digest of BBa_R0080 using SpeI and PstI
- Reaction mixture composition:
20 μl purified plasmid DNA product 0.5 μl SpeI (Fermentas) 1 μl PstI (Fermentas) 5 μl Buffer Tango (Fermentas) 24 μl MQ water
Task 3:
Methods:
- Ligation mixtures composition:
15 μl digested cro (or E0022) 8 μl digested vector 3 μl Tango buffer(Fermentas) 3 μl dNTPs mixture (EurX, concentration 5 mM) 1 μl ligase T4 (Fermentas)
- Duration of ligation was about 18 hours; reaction was conducted in 19 °c (approximately).
- In the next step ligated samples were thermally inactivated via heating in 80 °c for 20 minutes
Task 4:
- Prepare the bacterial cultures for isolation of on pSB1A3 plasmid containing:
April M T W T F S S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 May M T W T F S S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 June M T W T F S S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 July M T W T F S S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 August M T W T F S S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 September M T W T F S S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 October M T W T F S S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
- PCR programme: