Team:Warsaw/Calendar-Main/21 April 2009
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+ | <html> | ||
- | + | <h3>Cloning of hly gene into pKSII+ vector</h3> | |
- | <h3> | + | <h4><font color="black">Kamil</font></h4> |
- | <h4>Kamil | + | <br /> |
- | + | <p>Tasks:</p> | |
- | <img src="https:// | + | <ul> |
+ | <li>Amplification of hly</li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <li>Measurement of concentration of isolates from Yersinia and Listeria</li> | ||
+ | </ul> | ||
+ | <br /> | ||
+ | <p>Methods:</p> | ||
+ | <ul> | ||
+ | <li> | ||
+ | PCR mixture's composition: | ||
+ | <pre>2ul pfu buffer (Fermentas) | ||
+ | 1ul MgSO4 (Fermentas) | ||
+ | 0,5ul primers | ||
+ | 1,5ul dNTPs (10 mM, | ||
+ | 01,ul pfu polymerase (Fermentas) | ||
+ | 1ul template DNA from Listeria</pre> | ||
+ | </ul></li> | ||
+ | <p>Solution was topped up with H2O to 20ul.</p> | ||
+ | </p> | ||
+ | <ul> | ||
+ | <li>PCR program:</li> | ||
+ | <p>hly</p><pre align="left">300s 95°C <br/> (30s 95°C, 35s 42°C, 150s 72°C)x2 <br/> (30s 95°C, 35s 47°C, 150s 72°C)x28 <br/> 600s 72°C <br/> ~ 4°C</pre> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <li>Electrophoretic separation on 1% agarose gel</li> | ||
+ | <li>Measurement of concentration of isolates on NanoDrop</li> | ||
+ | </ul> | ||
+ | <br/> | ||
+ | <p>Results:</p> | ||
+ | <img src="https://static.igem.org/mediawiki/2009/7/76/2009.04.21_-_PCR_hly_opisany.jpg"/> | ||
<var> | <var> | ||
- | + | <ul> | |
- | + | <li>Gel (from left)</li> | |
- | + | </ul> | |
- | + | <ol> | |
+ | <li>GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li> | ||
+ | <li>hly</li> | ||
+ | <li>hly control -</li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <li>Measurement from NanoDrop: | ||
+ | + Yersinia - 29,6 ng/ul | ||
+ | + Listeria - 50,7 ng/ul</li> | ||
+ | </ul> | ||
+ | </var> | ||
+ | <br/> | ||
+ | <p>Conclusions:</p> | ||
+ | <ul> | ||
+ | <li>200ng of DNA should be used on sample</li> | ||
+ | <li>Time of anealing should be prolonged to 45s</li> | ||
+ | <li>Concentration of Mg should be increased</li> | ||
+ | </ul> | ||
+ | <br/> | ||
+ | </html> | ||
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{{WarNotebookEnd}} | {{WarNotebookEnd}} |
Latest revision as of 14:50, 17 September 2009
Cloning of hly gene into pKSII+ vector
Kamil
Tasks:
- Amplification of hly
- Measurement of concentration of isolates from Yersinia and Listeria
Methods:
-
PCR mixture's composition:
2ul pfu buffer (Fermentas) 1ul MgSO4 (Fermentas) 0,5ul primers 1,5ul dNTPs (10 mM, 01,ul pfu polymerase (Fermentas) 1ul template DNA from Listeria
Solution was topped up with H2O to 20ul.
- PCR program:
hly
300s 95°C
(30s 95°C, 35s 42°C, 150s 72°C)x2
(30s 95°C, 35s 47°C, 150s 72°C)x28
600s 72°C
~ 4°C
- Electrophoretic separation on 1% agarose gel
- Measurement of concentration of isolates on NanoDrop
Results:
- Gel (from left)
- GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
- hly
- hly control -
- Measurement from NanoDrop: + Yersinia - 29,6 ng/ul + Listeria - 50,7 ng/ul
Conclusions:
- 200ng of DNA should be used on sample
- Time of anealing should be prolonged to 45s
- Concentration of Mg should be increased
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