Team:Freiburg/Notebook

From 2012.igem.org

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__NOTOC__
__NOTOC__
<!--- The Mission, Experiments --->
<!--- The Mission, Experiments --->
-
=<span style="color:#2244AA"> Notebook =
+
=Notebook =
 +
----
 +
<br>
 +
[[File:notebooksymbolT.png|center|180px|link=]]
 +
<br>
 +
== 04/06 - 10/06 ==
-
== week 06/04/12 - 06/10/12 ==
+
:* Golden-Gate-Cloning-Reaction (GGC) to assemble a 12-repeat-TAL-TF, that recognizes the sequence TACATTGGACCTAT
-
• Golden-Gate-Cloning-Reaction (GGC) to assemble a 12-repeat-TAL-TF, that recognizes the sequence TACATTGGACCTAT --> protocol: sanjana et al. (first hexamers...)
+
:* transformation: GGC-reaction
-
• transformation: GGC-reaction
+
-
• making aliquots of ordered GGC-Primers (freiGEM-method)
+
-
• making aliquots of ordered, i.e. synthesized, direpeats
+
-
• extension-PCR of direpeats with ordered freiGEM-GGC-
+
-
• 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
+
-
== week 06/11/12 - 06/16/12 ==
+
:* making aliquots of ordered GGC-Primers (freiGEM-method)
-
• optimizing PCR conditions for extension of direpeats
+
:* making aliquots of ordered, i.e. synthesized, direpeats  
-
• redoing GGC-reaction --> protocol: sanjana et al.
+
-
• transformation of redone GGC-reaction
+
-
• GGC á la freiGEM --> transformation
+
-
• testing of iGEM-distribution kit
+
-
==  week 06/17/12 - 06/23/12 ==
+
:* extension-PCR of direpeats in order to produce diefferent restriction sites for correct location of direpeats
-
• cloning of direpeats into pJET 1.2 vector-system
+
:* PCR-Purification of extension-PCR
-
• colony-PCR of freiGEM-GGC product
+
-
• place sequencing order for freiGEM-GGC
+
-
== week 06/24/12 - 07/01/12 ==
+
:* 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 of freiGEM-GGC under various conditions
+
:* optimizing PCR conditions for extension of direpeats
-
• 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)
+
-
==  week 07/02/12 - 07/08/12 ==
+
:* redoing GGC-reaction
-
• extension-PCR with all 96 direpeats on one well-plate
+
:* transformation of redone GGC-reaction
-
• transformation
+
-
• PCR-amplification of all 4 iGEM-backbones --> testing different conditions
+
-
• making bacteria competent for transformation
+
-
==  week 07/09/12 - 07/15/12 ==
+
:* GGC á la freiGEM --> transformation
-
• digest of exDirepeats with XbaI and PstI
+
:* testing of iGEM-distribution kit
-
• nanodrop of exDirepeats
+
<br>
-
• ligation of exDirepeats in psB1C3 vector backbone and then transformation
+
==  17/06 - 23/06 ==
-
• colony-PCR, gel run, making cultures
+
-
• miniprep of some of the 96 exDirepeats
+
-
==  week 07/16/12 - 07/22/12 ==
+
:* cloning of direpeats into pJET 1.2 vector-system
-
• Transformation of synthesis products (Dnmt, hyperactive Gin, Gin L7C7, Tn3 and TAL-ORF) into DH10B-strain competent cells
+
:* colony-PCR of freiGEM-GGC product
-
• 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
+
-
==  week 23/07/12 - 07/29/12 ==
+
:* place sequencing order for freiGEM-GGC
 +
<br>
 +
==  24/06 - 01/07 ==
-
==  week 07/30/12 - 08/05/12 ==
+
:* optimizing of freiGEM-GGC under various conditions
-
• GGC to produce freiGEM-Mammo-Brick with CMV-promotor, Puromycin-resistance and postORF and TALEN-NG-vector backbone
+
:* transformation of pJET-direpeats into bacteria
-
• to do so a extension-PCR on the parts was done
+
-
• CMV-Promotor was taken out of iGEM Distribution Kit 2012
+
-
==  week 08/06/12 - 08/12/12 ==
+
:* using the iGEM distribution kit: Bba_A12564 (CMV+Luc) --> reporter-plasmid for DNA-methyl-transferase testing
-
• mutagenesis-PCR of pcb1c3 vector backbone to eliminate restriction site for BsmBI
+
:* Miniprep of GGC-transformation (1 successful)
-
• repeat of GGC to get MammoBrick
+
-
• gel-run of mutagenesis-PCR of psb1c3
+
-
==  week 08/13/12 - 08/19/12 ==
+
<br>
 +
==  02/07 - 08/07 ==
 +
:* extension-PCR with all 96 direpeats on one well-plate
 +
:* transformation
-
{|align="center"
+
:* PCR-amplification of all 4 iGEM-backbones --> testing different conditions
-
|[[Image:smallbarrev.png|960px|no frame]]
+
 
-
|}
+
:* 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





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