Team:Freiburg/Notebook

From 2012.igem.org

(Difference between revisions)
(Prototype team page)
 
(35 intermediate revisions not shown)
Line 1: Line 1:
-
<!-- *** What falls between these lines is the Alert Box!  You can remove it from your pages once you have read and understood the alert *** -->
+
{{Template:Team:Freiburg}}
 +
__NOTOC__
 +
<!--- The Mission, Experiments --->
 +
=Notebook =
 +
----
 +
<br>
 +
[[File:notebooksymbolT.png|center|180px|link=]]
 +
<br>
 +
== 04/06 - 10/06 ==
-
<html>
+
:* Golden-Gate-Cloning-Reaction (GGC) to assemble a 12-repeat-TAL-TF, that recognizes the sequence TACATTGGACCTAT
-
<div id="box" style="width: 700px; margin-left: 137px; padding: 5px; border: 3px solid #000; background-color: #fe2b33;">
+
-
<div id="template" style="text-align: center; font-weight: bold; font-size: large; color: #f6f6f6; padding: 5px;">
+
-
This is a template page. READ THESE INSTRUCTIONS.
+
-
</div>
+
-
<div id="instructions" style="text-align: center; font-weight: normal; font-size: small; color: #f6f6f6; padding: 5px;">
+
-
You are provided with this team page template with which to start the iGEM season.  You may choose to personalize it to fit your team but keep the same "look." Or you may choose to take your team wiki to a different level and design your own wiki.  You can find some examples <a href="https://2008.igem.org/Help:Template/Examples">HERE</a>.
+
-
</div>
+
-
<div id="warning" style="text-align: center; font-weight: bold; font-size: small; color: #f6f6f6; padding: 5px;">
+
-
You <strong>MUST</strong>  have all of the pages listed in the menu below with the names specified.  PLEASE keep all of your pages within your teams namespace. 
+
-
</div>
+
-
</div>
+
-
</html>
+
-
<!-- *** End of the alert box *** -->
+
:* transformation: GGC-reaction
-
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"
+
:* making aliquots of ordered GGC-Primers (freiGEM-method)
-
!align="center"|[[Team:Freiburg|Home]]
+
-
!align="center"|[[Team:Freiburg/Team|Team]]
+
-
!align="center"|[https://igem.org/Team.cgi?year=2012&team_name=Freiburg Official Team Profile]
+
-
!align="center"|[[Team:Freiburg/Project|Project]]
+
-
!align="center"|[[Team:Freiburg/Parts|Parts Submitted to the Registry]]
+
-
!align="center"|[[Team:Freiburg/Modeling|Modeling]]
+
-
!align="center"|[[Team:Freiburg/Notebook|Notebook]]
+
-
!align="center"|[[Team:Freiburg/Safety|Safety]]
+
-
!align="center"|[[Team:Freiburg/Attributions|Attributions]]
+
-
|}
+
 +
:* making aliquots of ordered, i.e. synthesized, direpeats
 +
:* extension-PCR of direpeats in order to produce diefferent restriction sites for correct location of direpeats
-
You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer.  It is a very useful organizational tool as well.
+
:* PCR-Purification of extension-PCR
 +
 
 +
:* mutagenesis-PCR of Zhang-plasmid to remove restriction sites (SpeI, PstI, EcoRI, XbaI) --> making the plasmid work with iGEM-standard
 +
<br>
 +
== 11/06 - 16/06 ==
 +
 
 +
:* optimizing PCR conditions for extension of direpeats
 +
 
 +
:* redoing GGC-reaction
 +
 
 +
:* transformation of redone GGC-reaction
 +
 
 +
:* GGC á la freiGEM --> transformation
 +
 
 +
:* testing of iGEM-distribution kit
 +
<br>
 +
==  17/06 - 23/06 ==
 +
 
 +
:* cloning of direpeats into pJET 1.2 vector-system
 +
 
 +
:* colony-PCR of freiGEM-GGC product
 +
 
 +
:* place sequencing order for freiGEM-GGC
 +
 
 +
<br>
 +
==  24/06 - 01/07 ==
 +
 
 +
:* optimizing of freiGEM-GGC under various conditions
 +
 
 +
:* transformation of pJET-direpeats into bacteria
 +
 
 +
:* using the iGEM distribution kit: Bba_A12564 (CMV+Luc) --> reporter-plasmid for DNA-methyl-transferase testing
 +
 
 +
:* Miniprep of GGC-transformation (1 successful)
 +
 
 +
<br>
 +
==  02/07 - 08/07 ==
 +
 
 +
:* extension-PCR with all 96 direpeats on one well-plate
 +
 
 +
:* transformation
 +
 
 +
:* PCR-amplification of all 4 iGEM-backbones --> testing different conditions
 +
 
 +
:* making bacteria competent for transformation
 +
 
 +
<br>
 +
== 09/07 - 15/07 ==
 +
 
 +
:* digest of exDirepeats with XbaI and PstI
 +
 
 +
:* nanodrop of exDirepeats
 +
 
 +
:* ligation of exDirepeats in psB1C3 vector backbone and then transformation
 +
 
 +
:* colony-PCR, gel run, making cultures
 +
 
 +
:* miniprep of some of the 96 exDirepeats
 +
 
 +
<br>
 +
==  16/07 - 22/07 ==
 +
 
 +
:* transformation of synthesis products (Dnmt, hyperactive Gin, Gin L7C7, Tn3 and TAL-ORF) into DH10B-strain competent cells
 +
 
 +
:* amplification of psb1c3 vector backbone, then gel-run and gel-purification
 +
 
 +
:* picking of colonies (transformation of synthesis-products)
 +
 
 +
:* miniprep of synthesis-products
 +
 
 +
:* repeat of pcr-amplification of psb1c3 vector backbone
 +
 
 +
<br>
 +
== 30/07 - 05/08 ==
 +
 
 +
:* GGC to produce freiGEM-Mammo-Brick with CMV-promotor, Puromycin-resistance and postORF and TALEN-NG-vector backbone
 +
 
 +
:* to do so a extension-PCR on the parts was done
 +
 
 +
:* CMV-Promotor was taken out of iGEM Distribution Kit 2012
 +
 
 +
<br>
 +
== 06/08 - 12/08 ==
 +
 
 +
:* mutagenesis-PCR of pcb1c3 vector backbone to eliminate restriction site for BsmBI
 +
 
 +
:* repeat of GGC to get MammoBrick
 +
 
 +
:* gel-run of mutagenesis-PCR of psb1c3
 +
 
 +
<br>
 +
== 13/08 - 19/08 ==
 +
 
 +
:* repeating the assembly of MammoBrick vector containing CMV Promotor, PostORF-Part and Puromycin-ORF
 +
 
 +
:* repeating mutagenesis-PCR on vector backbone psb1c3
 +
 
 +
:* extPCR of TAL-ORF (synthesis-product) in order to produce a n-terminal kind of adapter to paste recombinase
 +
 
 +
:* producing an provisory mammalian expression vector --> cloning of TAL-ORF into mammalian vector backbone
 +
 
 +
:* to do so the vector and the insert have been digested and then ligated and transformed into dh10b e. coli strain bacteria
 +
 
 +
:* transformation of mutated vector backbone
 +
 
 +
:* colony pcr to test provisory mammalian expression vector
 +
 
 +
<br>
 +
== 20/08 - 26/08 ==
 +
   
 +
:* repeating mutagenesis pcr for vector backbone psb1c3 with another template
 +
 
 +
:* finding a new method for successful extension pcr and then digest and ligation into the psb1cr backbone
 +
 
 +
 
 +
<br>
 +
== 27/08 - 02/09 ==
 +
 
 +
:* finding out that CMV promotor taken out of the registry distribution kit is not good at all
 +
 
 +
:* trying to get another vector with cmv promotor and ordering new primer pair to amplify it
 +
 
 +
:* after finding a working method for direpeat extension, digestion and ligation into psb1c3 vector backbone we now started to execute it on our synthesized direpeats
 +
 
 +
 
 +
<br>
 +
== 03/09 - 09/09 ==
 +
 
 +
:* GGC-reaction in order to produce recombinase-linker-n-tal-orf construct
 +
 
 +
:* annealing of linker-part (was ordered as two single-strand primers)
 +
 
 +
:* after ggc-reaction was done a pcr-amplification on the new assembled part was executed
 +
 
 +
:* gel-run shows a very weak band at 2,7 kb for hyperactive gin recombinase
 +
 
 +
:* GGC-reaction in order to clone direpeats for a complete TAL-Domain into provisory mammalian expression vector
 +
 
 +
:* repeating the extextPCR on the recombinase-linker-n-tal-orf construct under different conditions after the first pcr reaction failed
 +
 
 +
:* amplification of mutated psb1c3 vector backbone
 +
 
 +
:* trying to clone Transcription Factor into provisory mammalian expression vector
 +
 
 +
:* repeating of GGC reaction in order to produce MammoBrick
 +
 +
:* still working on our toolkit, doing the final steps: sent them off for sequencing
 +
 
 +
<br>
 +
== 10/09 - 16/09 ==
 +
 
 +
:* doing the final steps in producing our toolkit: miniprep was done for the last three batches of direpeats, which can finally be sequenced on monday
 +
 
 +
:* we now have 75 successfully sequenced direpeats in the psb1c3 vector backbone, ready to send them off to igem headquarters
 +
 
 +
:* new working cmv promotor is now in psb1c3 vector backbone, too. we now have an improved, i.e. finally working, biobrick for the registry
 +
 
 +
 
 +
<br>
 +
<br>
 +
<br>
 +
[[#top|Back to top]]

Latest revision as of 18:29, 24 October 2012




Notebook


NotebooksymbolT.png


04/06 - 10/06

  • Golden-Gate-Cloning-Reaction (GGC) to assemble a 12-repeat-TAL-TF, that recognizes the sequence TACATTGGACCTAT
  • transformation: GGC-reaction
  • making aliquots of ordered GGC-Primers (freiGEM-method)
  • making aliquots of ordered, i.e. synthesized, direpeats
  • extension-PCR of direpeats in order to produce diefferent restriction sites for correct location of direpeats
  • PCR-Purification of extension-PCR
  • mutagenesis-PCR of Zhang-plasmid to remove restriction sites (SpeI, PstI, EcoRI, XbaI) --> making the plasmid work with iGEM-standard


11/06 - 16/06

  • optimizing PCR conditions for extension of direpeats
  • redoing GGC-reaction
  • transformation of redone GGC-reaction
  • GGC á la freiGEM --> transformation
  • testing of iGEM-distribution kit


17/06 - 23/06

  • cloning of direpeats into pJET 1.2 vector-system
  • colony-PCR of freiGEM-GGC product
  • place sequencing order for freiGEM-GGC


24/06 - 01/07

  • optimizing of freiGEM-GGC under various conditions
  • transformation of pJET-direpeats into bacteria
  • using the iGEM distribution kit: Bba_A12564 (CMV+Luc) --> reporter-plasmid for DNA-methyl-transferase testing
  • Miniprep of GGC-transformation (1 successful)


02/07 - 08/07

  • extension-PCR with all 96 direpeats on one well-plate
  • transformation
  • PCR-amplification of all 4 iGEM-backbones --> testing different conditions
  • making bacteria competent for transformation


09/07 - 15/07

  • digest of exDirepeats with XbaI and PstI
  • nanodrop of exDirepeats
  • ligation of exDirepeats in psB1C3 vector backbone and then transformation
  • colony-PCR, gel run, making cultures
  • miniprep of some of the 96 exDirepeats


16/07 - 22/07

  • transformation of synthesis products (Dnmt, hyperactive Gin, Gin L7C7, Tn3 and TAL-ORF) into DH10B-strain competent cells
  • amplification of psb1c3 vector backbone, then gel-run and gel-purification
  • picking of colonies (transformation of synthesis-products)
  • miniprep of synthesis-products
  • repeat of pcr-amplification of psb1c3 vector backbone


30/07 - 05/08

  • GGC to produce freiGEM-Mammo-Brick with CMV-promotor, Puromycin-resistance and postORF and TALEN-NG-vector backbone
  • to do so a extension-PCR on the parts was done
  • CMV-Promotor was taken out of iGEM Distribution Kit 2012


06/08 - 12/08

  • mutagenesis-PCR of pcb1c3 vector backbone to eliminate restriction site for BsmBI
  • repeat of GGC to get MammoBrick
  • gel-run of mutagenesis-PCR of psb1c3


13/08 - 19/08

  • repeating the assembly of MammoBrick vector containing CMV Promotor, PostORF-Part and Puromycin-ORF
  • repeating mutagenesis-PCR on vector backbone psb1c3
  • extPCR of TAL-ORF (synthesis-product) in order to produce a n-terminal kind of adapter to paste recombinase
  • producing an provisory mammalian expression vector --> cloning of TAL-ORF into mammalian vector backbone
  • to do so the vector and the insert have been digested and then ligated and transformed into dh10b e. coli strain bacteria
  • transformation of mutated vector backbone
  • colony pcr to test provisory mammalian expression vector


20/08 - 26/08

  • repeating mutagenesis pcr for vector backbone psb1c3 with another template
  • finding a new method for successful extension pcr and then digest and ligation into the psb1cr backbone



27/08 - 02/09

  • finding out that CMV promotor taken out of the registry distribution kit is not good at all
  • trying to get another vector with cmv promotor and ordering new primer pair to amplify it
  • after finding a working method for direpeat extension, digestion and ligation into psb1c3 vector backbone we now started to execute it on our synthesized direpeats



03/09 - 09/09

  • GGC-reaction in order to produce recombinase-linker-n-tal-orf construct
  • annealing of linker-part (was ordered as two single-strand primers)
  • after ggc-reaction was done a pcr-amplification on the new assembled part was executed
  • gel-run shows a very weak band at 2,7 kb for hyperactive gin recombinase
  • GGC-reaction in order to clone direpeats for a complete TAL-Domain into provisory mammalian expression vector
  • repeating the extextPCR on the recombinase-linker-n-tal-orf construct under different conditions after the first pcr reaction failed
  • amplification of mutated psb1c3 vector backbone
  • trying to clone Transcription Factor into provisory mammalian expression vector
  • repeating of GGC reaction in order to produce MammoBrick
  • still working on our toolkit, doing the final steps: sent them off for sequencing


10/09 - 16/09

  • doing the final steps in producing our toolkit: miniprep was done for the last three batches of direpeats, which can finally be sequenced on monday
  • we now have 75 successfully sequenced direpeats in the psb1c3 vector backbone, ready to send them off to igem headquarters
  • new working cmv promotor is now in psb1c3 vector backbone, too. we now have an improved, i.e. finally working, biobrick for the registry





Back to top

Retrieved from "http://2012.igem.org/Team:Freiburg/Notebook"