"
Team:Warsaw/Calendar-Main/7 July 2009
From 2009.igem.org
(Difference between revisions)
| Line 7: | Line 7: | ||
<p>Tasks:</p> | <p>Tasks:</p> | ||
<ul> | <ul> | ||
| - | <li>Plasmid assembly | + | <li>Plasmid assembly |
</ul> | </ul> | ||
<br /> | <br /> | ||
| Line 16: | Line 16: | ||
| - | <p>UPDATE: No luck this time, back to the drawing board... | + | <p>UPDATE: No luck this time, back to the drawing board...</p> |
| + | </html> | ||
<h3>Yet another attempt at obtaining a specyific Llo PCR product for fusion with the secretion signal</h3> | <h3>Yet another attempt at obtaining a specyific Llo PCR product for fusion with the secretion signal</h3> | ||
| - | + | ||
<h4>Kuba</h4> | <h4>Kuba</h4> | ||
<br /> | <br /> | ||
| Line 29: | Line 30: | ||
<ul> | <ul> | ||
<li><p></li> | <li><p></li> | ||
| - | |||
<br /> | <br /> | ||
| - | + | Methods: | |
<ul><li><p>PCR mixture's composition:</p> 2,5ul pfu buffer (Fermentas), 2,5ul MgSO4 (Fermentas), 1,5ul primers, 1,5ul dNTPs (10 mM), 0,5ul pfu turbo polymerase, 1ul template DNA from Listeria, solution was topped up with H2O to 25ul. | <ul><li><p>PCR mixture's composition:</p> 2,5ul pfu buffer (Fermentas), 2,5ul MgSO4 (Fermentas), 1,5ul primers, 1,5ul dNTPs (10 mM), 0,5ul pfu turbo polymerase, 1ul template DNA from Listeria, solution was topped up with H2O to 25ul. | ||
| Line 39: | Line 39: | ||
<ul> | <ul> | ||
<li>PCR programs:</li> | <li>PCR programs:</li> | ||
| - | + | <pre align="middle"> 4min 95°C | |
| + | (30s 95°C, 40s 41°C, 1min40s 72°C)x3 | ||
| + | (30s 95°C, 40s 44°C, 1min40s 72°C)x28 10min 72°C | ||
| + | ~ 7°C</pre> | ||
| - | |||
</ul> | </ul> | ||
<ul> | <ul> | ||
| Line 48: | Line 50: | ||
<br /> | <br /> | ||
<p>Results:</p> | <p>Results:</p> | ||
| - | + | https://static.igem.org/mediawiki/2009/c/cd/Reamplifikacja_llo.JPG | |
<var> | <var> | ||
<ul> | <ul> | ||
| Line 63: | Line 65: | ||
<p>no siginificant amounts of desired product were obtained | <p>no siginificant amounts of desired product were obtained | ||
| - | |||
Revision as of 21:30, 8 July 2009
 |  |
 |
  |
 |
|
Yet another insertion of the mgtc gene into the pKSII+ plasmid
Kamil
Tasks:
- Plasmid assembly
Methods:
The ligation mix was prepared as follows: 1ul plasmid, 3ul gene, 1ul ligation buffer B (Fermentas), 1ul T4 DNA ligase (Fermentas), 1ul 30% PEG, 1ul 10mM ATP, the solution was topped up with H2O to the final volume of 10 ul. The ligation was carried out in 37°C for 1h and then inactivated for 15min. at 65°C.
A 200ul batch of chemocompetent bacteria was transformed with 3ul of ligation mix and incubated on petri dishes containing LB medium supplemented with ampicilin, X-gal and IPTG.
</li>
Methods:- <p>PCR mixture's composition:</p> 2,5ul pfu buffer (Fermentas), 2,5ul MgSO4 (Fermentas), 1,5ul primers, 1,5ul dNTPs (10 mM), 0,5ul pfu turbo polymerase, 1ul template DNA from Listeria, solution was topped up with H2O to 25ul. <p>One of the samples also contained 1 ul of DMSO</p>
- PCR programs:
4min 95°C (30s 95°C, 40s 41°C, 1min40s 72°C)x3 (30s 95°C, 40s 44°C, 1min40s 72°C)x28 10min 72°C ~ 7°C
- Electrophoretic separation on 1% agarose gel
- Gel (from left)
- GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
- sample no.1 (1ul of 100-fold diluted template)
- sample no. 2 (2ul of 100-fold diluted template)
- sample no. 3 (1ul of template)
UPDATE: No luck this time, back to the drawing board...
Yet another attempt at obtaining a specyific Llo PCR product for fusion with the secretion signal
Kuba
PCR was preformed on a product of classical biobrick-format Llo (reamplification)
three reactions were preformed, each with increasing concentration of template
no siginificant amounts of desired product were obtained
|
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
