"
Team:Warsaw/Calendar-Main/15 April 2009
From 2009.igem.org
(Difference between revisions)
| (10 intermediate revisions not shown) | |||
| Line 4: | Line 4: | ||
| - | <h3> | + | <h3>Cloning of the mgtc promoter into the pKSII+ plasmid</h3> |
| - | <h4>Kamil</h4> | + | <h4><font color="black">Kamil</font></h4> |
<br /> | <br /> | ||
<p>Tasks:</p> | <p>Tasks:</p> | ||
| Line 14: | Line 14: | ||
<p>Methods:</p> | <p>Methods:</p> | ||
<ul> | <ul> | ||
| - | <li><p>PCR mixture's composition:</p> 2ul buffer | + | <li><p>PCR mixture's composition:</p> |
| + | <pre> | ||
| + | 2ul buffer | ||
| + | 1ul MgCl2 | ||
| + | 0,5ul primers | ||
| + | 1,5ul dNTPs (10 mM) | ||
| + | 01,ul polymerase | ||
| + | </pre> | ||
| + | <p>Solution was topped up with H2O to 20ul.</p> | ||
<p>2 repeats of every sample were made.</p> | <p>2 repeats of every sample were made.</p> | ||
</li> | </li> | ||
| Line 20: | Line 28: | ||
<ul> | <ul> | ||
<li>PCR programs:</li> | <li>PCR programs:</li> | ||
| - | <pre align="left" | + | <p>mgtc</p><pre align="left">90s 95°C <br/> (30s 95°C, 35s 48°C, 60s 72°C)x2 <br/> (30s 95°C, 35s 58°C, 60s 72°C)x28 <br/> 600s 72°C <br/> ~ 4°C</pre> |
| - | <pre align="left" | + | <p>hly</p><pre align="left">90s 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/> ~ 10°C</pre> |
</ul> | </ul> | ||
<ul> | <ul> | ||
| Line 30: | Line 38: | ||
<img src="https://static.igem.org/mediawiki/2009/7/70/2009.04.15_-_PCR_mgtc_i_hly_opisane.jpg"/> | <img src="https://static.igem.org/mediawiki/2009/7/70/2009.04.15_-_PCR_mgtc_i_hly_opisane.jpg"/> | ||
<var> | <var> | ||
| - | < | + | <ul> |
| + | <li>Gel (from left)</li> | ||
| + | </ul> | ||
<ol> | <ol> | ||
<li>GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li> | <li>GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li> | ||
<li>mgtc 1 repeat</li> | <li>mgtc 1 repeat</li> | ||
<li>mgtc 2 repeat</li> | <li>mgtc 2 repeat</li> | ||
| - | <li>mgtc | + | <li>mgtc control -</li> |
<li>hly 1 repeat</li> | <li>hly 1 repeat</li> | ||
<li>hly 2 repeat</li> | <li>hly 2 repeat</li> | ||
| - | <li>hly | + | <li>hly control -</li> |
</ol> | </ol> | ||
| Line 44: | Line 54: | ||
<br /> | <br /> | ||
<p>Notes:</p> | <p>Notes:</p> | ||
| - | < | + | <ul> |
| - | < | + | <li>Inappropriate template DNA was used for mgtc (ganomic DNA from Yersinia instead of genomic DNA from Listeria).</li> |
| + | </ul> | ||
| + | <br /> | ||
</html> | </html> | ||
Latest revision as of 14:41, 17 September 2009
 |  |
 |
  |
 |
|
Cloning of the mgtc promoter into the pKSII+ plasmid
Kamil
Tasks:
- Amplification of mgtc and hly
Methods:
PCR mixture's composition:
2ul buffer 1ul MgCl2 0,5ul primers 1,5ul dNTPs (10 mM) 01,ul polymerase
Solution was topped up with H2O to 20ul.
2 repeats of every sample were made.
- PCR programs:
mgtc
90s 95°C
(30s 95°C, 35s 48°C, 60s 72°C)x2
(30s 95°C, 35s 58°C, 60s 72°C)x28
600s 72°C
~ 4°C
hly
90s 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
~ 10°C
- Electrophoretic separation on 1% agarose gel
Results:
- Gel (from left)
- GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
- mgtc 1 repeat
- mgtc 2 repeat
- mgtc control -
- hly 1 repeat
- hly 2 repeat
- hly control -
Notes:
- Inappropriate template DNA was used for mgtc (ganomic DNA from Yersinia instead of genomic DNA from Listeria).
|
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
