Team:Warsaw/Calendar-Main/3 September 2009

From 2009.igem.org

(Difference between revisions)
(New page: {{WarNotebook}} <!-- do not edit above me! --> ===<div style="text-align: center;">Acquiring the internaline A gene from ''Listeria monocytogenes'' st. EDG-e genome </div>=== '''Jarek'''...)
 
(7 intermediate revisions not shown)
Line 16: Line 16:
 +
===<div style="text-align: center;"><h3>Preparation of glycerol stocks</h3></div>===
 +
'''<h4>Monika</h4>'''
 +
 +
 +
*Preparation of glycerol stocks following the prodcedure described [https://2009.igem.org/Team:Warsaw/protocols#Preparation_of_glycerol_stocks <span style="color: blue;">here</span>]
 +
 +
 +
pAraC + GFP
 +
 +
plac + RBS + cI + ter
 +
 +
plac + RBS + cI + RBS + GFP + ter
 +
 +
J07037
 +
 +
plac
 +
 +
pSB6A1
 +
 +
 +
<h3><div style="text-align: center;">Assembly of endosomal detection operon</div></h3>
 +
<h4>Marcin</h4>
 +
 +
Task 1:
 +
* Isolate the plasmids containing  following construct:
 +
* [http://partsregistry.org/Part:BBa_K177035<span style="color: black">BBa_K177035</span>] on [http://partsregistry.org/Part:pSB1A3<span style="color: black">pSB1A3</span>] plasmid
 +
Methods:
 +
Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described [http://aabiot.com/products/dna_purification/plasmid_dna/plasmid_mini/protocol_plasmid_mini.pdf here]
 +
 +
Task 2:
 +
* Digestion of plasmids previously described constructs
 +
Methods:
 +
* Reaction mixture composition:
 +
<pre>
 +
20 μl purified plasmid DNA product
 +
1 μl XbaI (Fermentas)
 +
1 μl PstI (Fermentas)
 +
2 μl Buffer Tango (Fermentas)
 +
15 μl MQ water
 +
</pre>
 +
* Digest was performed about six hours and subsequently thermal inactivated
 +
 +
Task 3:
 +
* Gel-outs of construct described in Task 2
 +
Methods:
 +
* All gel-outs were performed using the EurX gel-out kit according to the manual
 +
 +
Task 4:
 +
 
 +
*  Ligation of following constructs:
 +
** [http://partsregistry.org/Part:BBa_K177037<span style="color: black">BBa_K177037</span>] on [http://partsregistry.org/Part:pSB1A3<span style="color: black">pSB1A3</span>] plasmid
 +
** cro CDS on [http://partsregistry.org/Part:pSB1A3<span style="color: black">pSB1A3</span>]
 +
** Bax CDS on [http://partsregistry.org/Part:pSB1A3<span style="color: black">pSB1A3</span>]
 +
Methods:
 +
* Reaction mixture composition:
 +
<pre>
 +
10 μl digested insert
 +
8 μl digested vector
 +
2 μl Tango buffer(Fermentas)
 +
3 μl dNTPs mixture (EurX, concentration 5 mM)
 +
1 μl ligase T4 (Fermentas)
 +
</pre>
 +
* Reaction were carried out 12 hours and subsequently thermally inactivated.
 +
 +
<html>
 +
<h3><div style="text-align: center;">Cloning of the mgtc promoter into the pSB1A3 plasmid</div></h3>
 +
<h4>Kamil</h4>
 +
<br />
 +
<p>Tasks:</p>
 +
<ul>
 +
<li>Plasmid isolation</li>
 +
<li>Control digest</li>
 +
</ul>
 +
<br />
 +
<p>Methods:</p>
 +
<ul>
 +
<li>Plasmids from 3 ml of overnight culture were isolated using the Plasmid Mini kit (A&A Biotechnology).</li>
 +
<li>The digest mix was prepared as follows:
 +
<p><pre>20&mu;l isolated plasmid<br/>3&mu;l Buffer O (Fermentas)<br/>1&mu;l PstI enzyme<br/>1&mu;l EcoRI enzyme<br/>5&mu;l H<sub>2</sub>O</pre></p></li>
 +
<li>The digest was carried out for 3h in 37&deg;C and then inactivated in 80&deg;C for 15min.</li>
 +
<li>The results were visualised on 1% agarose gel</li>
 +
</ul>
 +
<br />
 +
<p>Results:</p>
 +
<ul>
 +
<img src="https://static.igem.org/mediawiki/2009/6/65/2009.09.03.jpg">
 +
<li>First four bands (besides the the size ruler) show empty plasmids.</li>
 +
</ul>
 +
<br/>
 +
<p>Conclusions:</p>
 +
<ul>
 +
<li>Respawn and try again in... 3 days.</li>
 +
</ul>
 +
<br/>
 +
 +
 +
<h3><div style="text-align: center;">Cloning of the cro-box into the pSB1A3 plasmid</div></h3>
 +
<h4>Kamil</h4>
 +
<br />
 +
<p>Tasks:</p>
 +
<ul>
 +
<li>Plasmid isolation</li>
 +
<li>Control digest</li>
 +
</ul>
 +
<br />
 +
<p>Methods:</p>
 +
<ul>
 +
<li>Plasmids from 3 ml of overnight culture were isolated using the Plasmid Mini kit (A&A Biotechnology).</li>
 +
<li>The digest mix was prepared as follows:
 +
<p><pre>20&mu;l isolated plasmid<br/>3&mu;l Buffer O (Fermentas)<br/>1&mu;l PstI enzyme<br/>1&mu;l EcoRI enzyme<br/>5&mu;l H<sub>2</sub>O</pre></p></li>
 +
<li>The digest was carried out for 3h in 37&deg;C and then inactivated in 80&deg;C for 15min.</li>
 +
<li>The results were visualised on 1% agarose gel</li>
 +
</ul>
 +
<br />
 +
<p>Results:</p>
 +
<ul>
 +
<img src="https://static.igem.org/mediawiki/2009/6/65/2009.09.03.jpg">
 +
<li>Final five bands show empty plasmids.</li>
 +
</ul>
 +
<br/>
 +
<p>Conclusions:</p>
 +
<ul>
 +
<li>Respawn and try again in... 3 days.</li>
 +
</ul>
 +
<br/>
 +
<h3><div style="text-align: center;">Cotransformation to obtain cells with following plasmid combinations [<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012">K177012</a>,<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177011">K177011</a>][<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012">K177012</a>,<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177038">K177038</a>][<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177033">K177033</a>,<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177011">K177011</a>][<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177033">K177033</a>,<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177038">K177038</a>]
 +
<h4>Ania</h4>
 +
<br/>
 +
<p>Tasks:</p>
 +
<ul>
 +
<li>
 +
</li>
 +
</ul>
 +
<br/>
 +
</html>

Latest revision as of 22:16, 20 September 2009


Previous day
return to main notebook page
Previous entry
next notebook entry

 


Contents

Acquiring the internaline A gene from Listeria monocytogenes st. EDG-e genome

Jarek

Tasks:

  • Digestion of PCR products, separetly with MboI and CaiI restriction endonucleases
  • Electrophoretical segregation of digested samples in 1,5% agarose gel
  • Preparation of another PCR reaction with L.monocytogenes genome DNA


Results:

  • There were no visible products of digestion, even after long treatment with ethidium bromide. Propably the Clean-Up kit isn't working well. I'll try to acquire IntA gene with another PCR.


Preparation of glycerol stocks

Monika


  • Preparation of glycerol stocks following the prodcedure described here


pAraC + GFP

plac + RBS + cI + ter

plac + RBS + cI + RBS + GFP + ter

J07037

plac

pSB6A1


Assembly of endosomal detection operon

Marcin

Task 1:

Methods: Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described here

Task 2:

  • Digestion of plasmids previously described constructs

Methods:

  • Reaction mixture composition: 
20 μl purified plasmid DNA product
1 μl XbaI (Fermentas)
1 μl PstI (Fermentas) 
2 μl Buffer Tango (Fermentas)
15 μl MQ water
  • Digest was performed about six hours and subsequently thermal inactivated

Task 3:

  • Gel-outs of construct described in Task 2

Methods:

  • All gel-outs were performed using the EurX gel-out kit according to the manual

Task 4:

Methods:

  • Reaction mixture composition: 
10 μl digested insert
8 μl digested vector 
2 μl Tango buffer(Fermentas)
3 μl dNTPs mixture (EurX, concentration 5 mM) 
1 μl ligase T4 (Fermentas)
  • Reaction were carried out 12 hours and subsequently thermally inactivated.

Cloning of the mgtc promoter into the pSB1A3 plasmid

Kamil


Tasks:

  • Plasmid isolation
  • Control digest

Methods:

  • Plasmids from 3 ml of overnight culture were isolated using the Plasmid Mini kit (A&A Biotechnology).
  • The digest mix was prepared as follows: 

    20μl isolated plasmid
    3μl Buffer O (Fermentas)
    1μl PstI enzyme
    1μl EcoRI enzyme
    5μl H2O

  • The digest was carried out for 3h in 37°C and then inactivated in 80°C for 15min.
  • The results were visualised on 1% agarose gel

Results:

  • First four bands (besides the the size ruler) show empty plasmids.

Conclusions:

  • Respawn and try again in... 3 days.

Cloning of the cro-box into the pSB1A3 plasmid

Kamil


Tasks:

  • Plasmid isolation
  • Control digest

Methods:

  • Plasmids from 3 ml of overnight culture were isolated using the Plasmid Mini kit (A&A Biotechnology).
  • The digest mix was prepared as follows: 

    20μl isolated plasmid
    3μl Buffer O (Fermentas)
    1μl PstI enzyme
    1μl EcoRI enzyme
    5μl H2O

  • The digest was carried out for 3h in 37°C and then inactivated in 80°C for 15min.
  • The results were visualised on 1% agarose gel

Results:

  • Final five bands show empty plasmids.

Conclusions:

  • Respawn and try again in... 3 days.

Cotransformation to obtain cells with following plasmid combinations [K177012,K177011][K177012,K177038][K177033,K177011][K177033,K177038]

Ania


Tasks:




April
MTWTFSS
    1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30
May
MTWTFSS
        1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31
June
MTWTFSS
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30
July
MTWTFSS
    1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30 31
August
MTWTFSS
          1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31
September
MTWTFSS
  1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30
October
MTWTFSS
      1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 31
Retrieved from "http://2009.igem.org/Team:Warsaw/Calendar-Main/3_September_2009"