Team:IPN-UNAM-Mexico/Notebook/July

From 2009.igem.org

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For transformation we use the same method as follow.
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For transformation we used the following method:
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*Elusion: 3μl DNA from the well.
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*Elution: 3μl DNA from the well.
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*Put it on a Eppendorf Vial 1.5 ml with competent cells.
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*Put it on a 1.5 ml Eppendorf vial with competent cells.
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*Electoporate shock to transform and recovery in LB ampr 1 hour and a half.
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*Transformation was made by electroporation and recovery in LB media with Ampr for 1 hour and a half.
*Plate on petri dishes and incubate 17 hours.  
*Plate on petri dishes and incubate 17 hours.  

Revision as of 07:19, 21 October 2009


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Contents

07-July-2009

  • Digestion of the following biobricks were done with EcoRI (E), SpeI (S), and XbaI (X).
Biobrick Enzyme restriction
R0079 E, S
F1610 E, X
B0034 E, S
C0078 E, X
C0079 E, X
B0015 E, X


  • We propose this restrictions for standard assembly for the digestion we did a 17 hours incubation at 37°C.
  • Digest Mix:
EcoRI - SpeI Per reaction
Plasmidic DNA 3μl
Enzyme EcoRI 2μl
Enzyme SpeI 2μl
Buffer NBE 2μl
BSA 0.5μl
H2O 10.5μl
Total 20μl
EcoRI - XbaI Per reaction
Plasmidic DNA 3μl
Enzyme EcoRI 2μl
Enzyme XbaI 2μl
Buffer NBE 2μl
BSA 0.5μl
H20 10.5μl
Total 20μl


08-July-2009

  • We made the restrictions and put them on an agarose gel with 10 wells for 40 min.
  • We used 3μl DNA plus 2μl Buffer.
  • We didn't get any DNA from the gel so maybe the DNA volume is too low. We tried again with 20μl DNA plus 3μl Buffer
  • Run on only Plasmidic DNA but we have no DNA.


09-July-2009

  • Starting again, we extracted DNA from the folder plates R0079, C0178, C0179, C0060, B0034, I739001.
  • Left for 16 hours at 37°C.


10-July-2009

We didn't have any transformed cells so at this point we had to delay wetlab work and request 2009 catalog.



28-July-2009

After the 2009 catalog arrived we could resume the wetlab work because some of the biobricks we needed were not available on previous folders. First of all we tested them to be sure that the biobricks worked properly, for this we took a biobrick with RFP reporter (BBa_I3522), plated and incubated for 17 hours at 37ºC.



29-July-2009

The transformation was successful, so we are now sure that the biobricks and methods we used work correctly so the next step is to extract the biobricks we are going to use for our project which are the following:

Number Biobrick
2 BBa_R0079
3 BBa_F1610
4 BBa_K091146
5 BBa_K093005
6 BBa_K081016
7 BBa_EC840
8 BBa_K081009
9 BBa_R0051
10 BBa_J06800
11 BBa_Q04121
12 BBa_B0034
13 BBa_C0079
14 BBa_C0179
15 BBa_B0015
16 BBa_K081018
17 BBa_K116640

For transformation we used the following method:

  • Elution: 3μl DNA from the well.
  • Put it on a 1.5 ml Eppendorf vial with competent cells.
  • Transformation was made by electroporation and recovery in LB media with Ampr for 1 hour and a half.
  • Plate on petri dishes and incubate 17 hours.


31-July-2009

  • Plated Top 10 biobricks F1610, K091146, K081016, K081009, R0051, K081018 and incubate during 17 hours



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