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Team:Warsaw/Calendar-Main/22 April 2009
From 2009.igem.org
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| - | <h3> | + | <h3>Cloning of hly gene into pKSII+ vector</h3> |
| - | <h4> | + | <h4>Kama</h4> |
| - | + | ||
<p>Tasks:</p> | <p>Tasks:</p> | ||
<ul> | <ul> | ||
| - | <li>Amplification of | + | <li>Amplification of hly</li> |
</ul> | </ul> | ||
<br /> | <br /> | ||
<p>Methods:</p> | <p>Methods:</p> | ||
| - | <ul><li><p>PCR mixture's composition:</p> | + | <ul><li><p>PCR mixture's composition:</p> |
| - | <p> | + | <pre>2,5μl pfu buffer (Fermentas) |
| + | 2,5μl MgSO4 (Fermentas) | ||
| + | 1,5μl primers | ||
| + | 1,5μl dNTPs (10 mM) | ||
| + | 0,5μl pfu turbo polymerase <font color="green">(od Antka, ale KNGiE też powinna działać)</font> | ||
| + | 1μl template DNA from Listeria</pre> | ||
| + | <p>Solution was topped up with H2O to 25μl.</p> | ||
| + | <p>1 repeat of every sample was made (2 different programs).</p> | ||
| + | <p>Additionally for each sample was made version with 10∗ dilution of template.</p> | ||
</li> | </li> | ||
</ul> | </ul> | ||
<ul> | <ul> | ||
<li>PCR programs:</li> | <li>PCR programs:</li> | ||
| - | <p> | + | <p>hly</p><pre align="left">4min 95°C <br/> (30s 95°C, 35s 42°C, 1min20s 72°C)x3 <br/> (30s 95°C, 35s 47°C, 1min20s 72°C)x28 <br/> 10min 72°C <br/> ~ 7°C</pre> |
| - | + | <p>random program</p> | |
</ul> | </ul> | ||
<ul> | <ul> | ||
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<br /> | <br /> | ||
<p>Results:</p> | <p>Results:</p> | ||
| - | <img src="https://static.igem.org/mediawiki/2009/ | + | <img src="https://static.igem.org/mediawiki/2009/5/5e/2009.04.22_-_PCR_hly_opisany.jpg"/> |
<var> | <var> | ||
<ul> | <ul> | ||
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<ol> | <ol> | ||
<li>GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li> | <li>GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li> | ||
| - | <li> | + | <li>hly control -</li> |
| - | <li> | + | <li>hly</li> |
| - | <li> | + | <li>hly (10x diluted template)</li> |
| - | <li>hly | + | <li>hly control -*</li> |
| - | <li>hly | + | <li>hly*</li> |
| - | <li>hly | + | <li>hly (10x diluted template)*</li> |
| - | + | ||
</ol> | </ol> | ||
| + | <p>* random program</p> | ||
| + | <br/> | ||
| + | <p><b>hly was succesfully amplified<font color="green"> (termocykler z gradientem w pokoju 145)</font></b></p> | ||
</var> | </var> | ||
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Latest revision as of 14:52, 17 September 2009
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Cloning of hly gene into pKSII+ vector
Kama
Tasks:
- Amplification of hly
Methods:
PCR mixture's composition:
2,5μl pfu buffer (Fermentas) 2,5μl MgSO4 (Fermentas) 1,5μl primers 1,5μl dNTPs (10 mM) 0,5μl pfu turbo polymerase (od Antka, ale KNGiE też powinna działać) 1μl template DNA from Listeria
Solution was topped up with H2O to 25μl.
1 repeat of every sample was made (2 different programs).
Additionally for each sample was made version with 10∗ dilution of template.
- PCR programs:
hly
4min 95°C
(30s 95°C, 35s 42°C, 1min20s 72°C)x3
(30s 95°C, 35s 47°C, 1min20s 72°C)x28
10min 72°C
~ 7°C
random program
- Electrophoretic separation on 1% agarose gel
Results:
- Gel (from left)
- GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
- hly control -
- hly
- hly (10x diluted template)
- hly control -*
- hly*
- hly (10x diluted template)*
* random program
hly was succesfully amplified (termocykler z gradientem w pokoju 145)
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