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Team:Warsaw/Calendar-Main/21 April 2009
From 2009.igem.org
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| - | <h3> | + | <h3>Cloning of hly gene into pKSII+ vector</h3> |
| - | <h4><font color=" | + | <h4><font color="black">Kamil</font></h4> |
<br /> | <br /> | ||
<p>Tasks:</p> | <p>Tasks:</p> | ||
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<p>Methods:</p> | <p>Methods:</p> | ||
<ul> | <ul> | ||
| - | <li> | + | <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> | </p> | ||
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| - | |||
<ul> | <ul> | ||
<li>PCR program:</li> | <li>PCR program:</li> | ||
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<li>GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li> | <li>GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li> | ||
<li>hly</li> | <li>hly</li> | ||
| - | <li>hly | + | <li>hly control -</li> |
</ol> | </ol> | ||
<ul> | <ul> | ||
Latest revision as of 14:50, 17 September 2009
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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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