"
Team:Warsaw/Calendar-Main/22 April 2009
From 2009.igem.org
(Difference between revisions)
| (5 intermediate revisions not shown) | |||
| Line 2: | Line 2: | ||
<!-- do not edit above me! --> | <!-- do not edit above me! --> | ||
| - | <html | + | <html> |
| - | <h3> | + | <h3>Cloning of hly gene into pKSII+ vector</h3> |
<h4>Kama</h4> | <h4>Kama</h4> | ||
| - | |||
<p>Tasks:</p> | <p>Tasks:</p> | ||
<ul> | <ul> | ||
| Line 14: | Line 13: | ||
<br /> | <br /> | ||
<p>Methods:</p> | <p>Methods:</p> | ||
| - | <ul><li><p>PCR mixture's composition:</p> 2, | + | <ul><li><p>PCR mixture's composition:</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>1 repeat of every sample was made (2 different programs).</p> | ||
| - | <p>Additionally for each sample was made version with | + | <p>Additionally for each sample was made version with 10∗ dilution of template.</p> |
</li> | </li> | ||
</ul> | </ul> | ||
| Line 53: | Line 59: | ||
</html> | </html> | ||
| + | {{WarNotebookEnd}} | ||
Latest revision as of 14:52, 17 September 2009
 |  |
 |
  |
 |
|
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)
|
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
