From 2012.igem.org

(Difference between revisions)

Latest revision as of 18:29, 24 October 2012

Notebook

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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@@ Line 1: / Line 1: @@
 {{Template:Team:Freiburg}}
 __NOTOC__
-{|align="center"
+<!--- The Mission, Experiments --->
-|[[Image:smallbarfor.png|960px|no frame]]
+=Notebook =
-|}
+----
-{| style="color:#4F8DDE;background-color:#ffffff;" cellpadding="5" cellspacing="2" border="0" bordercolor="#fff" width="100%" align="left"
+<br>
-!align="center"|[[Team:Freiburg|<span style="color:#4F8DDE;">Home</span>]]
+[[File:notebooksymbolT.png|center|180px|link=]]
-!align="center"|[[Team:Freiburg/Team|<span style="color:#4F8DDE;">Team</span>]]
+<br>
-!align="center"|[https://igem.org/Team.cgi?year=2012&team_name=Freiburg<span style="color:#4F8DDE;">Official Team Profile</span>]
+== 04/06 - 10/06 ==
-!align="center"|[[Team:Freiburg/Project|<span style="color:#4F8DDE;">Project</span>]]
-!align="center"|[[Team:Freiburg/Parts|<span style="color:#4F8DDE;">Parts Submitted to the Registry</span>]]
-!align="center"|[[Team:Freiburg/Modeling|<span style="color:#4F8DDE;">Modeling</span>]]
-!align="center"|[[Team:Freiburg/Notebook|<span style="color:#4169E1;">Notebook</span>]]
-!align="center"|[[Team:Freiburg/Safety|<span style="color:#4F8DDE;">Safety</span>]]
-!align="center"|[[Team:Freiburg/Attributions|<span style="color:#4F8DDE;">Attributions</span>]]
-|}
-{|align="center"
-|[[Image:smallbarrev.png|960px|no frame]]
-|}
+:* 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
-<!--- The Mission, Experiments --->
+:* PCR-Purification of extension-PCR
-=<span style="color:#2244AA"> Notebook =
+:* 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
-{|align="justify"
-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.
-Coming soon...
-|}
-{|align="center"
+<br>
-|[[Image:smallbarrev.png|960px|no frame]]
+<br>
-|}
+<br>
+[[#top|Back to top]]