Team:Warsaw/Calendar-Main/3 September 2009

From 2009.igem.org

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<li>Respawn and try again in... 3 days.</li>
<li>Respawn and try again in... 3 days.</li>
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<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>]
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<h4>Ania</h4>
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<p>Tasks:</p>
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Revision as of 00:44, 10 September 2009


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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:

  • Isolate the plasmids containing following construct:
  • BBa_C0051 with BBa_B0032 on pSB1A3 plasmid

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

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:

  • [Nice gel picture of empty vectors here]

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:

  • [Nice gel picture of empty vectors here]

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
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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
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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
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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
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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
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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
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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