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	<!--- The Mission, Experiments --->		<!--- The Mission, Experiments --->
	=Notebook =		=Notebook =
		+	----
	<br>		<br>
-	== week 06/04/12 - 06/10/12 ==	+	[[File:notebooksymbolT.png|center|180px|link=]]
		+	<br>
		+	== 04/06 - 10/06 ==
-	• Golden-Gate-Cloning-Reaction (GGC) to assemble a 12-repeat-TAL-TF, that recognizes the sequence TACATTGGACCTAT --> protocol: sanjana et al. (first hexamers...)	+	:* Golden-Gate-Cloning-Reaction (GGC) to assemble a 12-repeat-TAL-TF, that recognizes the sequence TACATTGGACCTAT
-	• transformation: GGC-reaction	+	:* transformation: GGC-reaction
-	• making aliquots of ordered GGC-Primers (freiGEM-method)	+	:* making aliquots of ordered GGC-Primers (freiGEM-method)
-	• making aliquots of ordered, i.e. synthesized, direpeats	+	:* making aliquots of ordered, i.e. synthesized, direpeats
-	• extension-PCR of direpeats in order to produce diefferent restriction sites for correct location of direpeats	+	:* extension-PCR of direpeats in order to produce diefferent restriction sites for correct location of direpeats
-	• PCR-Purification of extension-PCR	+	:* 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	+	:* 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
-	== week 06/11/12 - 06/16/12 ==	+	:* redoing GGC-reaction
-	• optimizing PCR conditions for extension of direpeats	+	:* transformation of redone GGC-reaction
-	• redoing GGC-reaction --> protocol: sanjana et al.	+	:* GGC á la freiGEM --> transformation
-	• transformation of redone GGC-reaction	+	:* testing of iGEM-distribution kit
		+	<br>
		+	==  17/06 - 23/06 ==
-	• GGC á la freiGEM --> transformation	+	:* cloning of direpeats into pJET 1.2 vector-system
-	• testing of iGEM-distribution kit	+	:* colony-PCR of freiGEM-GGC product
		+	:* place sequencing order for freiGEM-GGC
-	==  week 06/17/12 - 06/23/12 ==	+	<br>
		+	==  24/06 - 01/07 ==
-	• cloning of direpeats into pJET 1.2 vector-system	+	:* optimizing of freiGEM-GGC under various conditions
-	• colony-PCR of freiGEM-GGC product	+	:* transformation of pJET-direpeats into bacteria
-	• place sequencing order for freiGEM-GGC	+	:* using the iGEM distribution kit: Bba_A12564 (CMV+Luc) --> reporter-plasmid for DNA-methyl-transferase testing
		+	:* Miniprep of GGC-transformation (1 successful)
-	==  week 06/24/12 - 07/01/12 ==	+	<br>
		+	==  02/07 - 08/07 ==
-	• optimizing of freiGEM-GGC under various conditions	+	:* extension-PCR with all 96 direpeats on one well-plate
-	• transformation of pJET-direpeats into bacteria	+	:* transformation
-	• using the iGEM distribution kit: Bba_A12564 (CMV+Luc) --> reporter-plasmid for DNA-methyl-transferase testing	+	:* PCR-amplification of all 4 iGEM-backbones --> testing different conditions
-	• Miniprep of GGC-transformation (1 successful)	+	:* making bacteria competent for transformation
		+	<br>
		+	== 09/07 - 15/07 ==
-	==  week 07/02/12 - 07/08/12 ==	+	:* digest of exDirepeats with XbaI and PstI
-	• extension-PCR with all 96 direpeats on one well-plate	+	:* nanodrop of exDirepeats
-	• transformation	+	:* ligation of exDirepeats in psB1C3 vector backbone and then transformation
-	• PCR-amplification of all 4 iGEM-backbones --> testing different conditions	+	:* colony-PCR, gel run, making cultures
-	• making bacteria competent for transformation	+	:* miniprep of some of the 96 exDirepeats
		+	<br>
		+	==  16/07 - 22/07 ==
-	==  week 07/09/12 - 07/15/12 ==	+	:* transformation of synthesis products (Dnmt, hyperactive Gin, Gin L7C7, Tn3 and TAL-ORF) into DH10B-strain competent cells
-	• digest of exDirepeats with XbaI and PstI	+	:* amplification of psb1c3 vector backbone, then gel-run and gel-purification
-	• nanodrop of exDirepeats	+	:* picking of colonies (transformation of synthesis-products)
-	• ligation of exDirepeats in psB1C3 vector backbone and then transformation	+	:* miniprep of synthesis-products
-	• colony-PCR, gel run, making cultures	+	:* repeat of pcr-amplification of psb1c3 vector backbone
-	• miniprep of some of the 96 exDirepeats	+	<br>
		+	== 30/07 - 05/08 ==
		+	:* GGC to produce freiGEM-Mammo-Brick with CMV-promotor, Puromycin-resistance and postORF and TALEN-NG-vector backbone
-	==  week 07/16/12 - 07/22/12 ==	+	:* to do so a extension-PCR on the parts was done
-	• Transformation of synthesis products (Dnmt, hyperactive Gin, Gin L7C7, Tn3 and TAL-ORF) into DH10B-strain competent cells	+	:* CMV-Promotor was taken out of iGEM Distribution Kit 2012
-	• amplification of psb1c3 vector backbone, then gel-run and gel-purification	+	<br>
		+	==  06/08 - 12/08 ==
-	• picking of colonies (transformation of synthesis-products)	+	:* mutagenesis-PCR of pcb1c3 vector backbone to eliminate restriction site for BsmBI
-	• miniprep of synthesis-products	+	:* repeat of GGC to get MammoBrick
-	• repeat of pcr-amplification of psb1c3 vector backbone	+	:* gel-run of mutagenesis-PCR of psb1c3
		+	<br>
		+	== 13/08 - 19/08 ==
-	==  week 07/30/12 - 08/05/12 ==	+	:* repeating the assembly of MammoBrick vector containing CMV Promotor, PostORF-Part and Puromycin-ORF
-	• GGC to produce freiGEM-Mammo-Brick with CMV-promotor, Puromycin-resistance and postORF and TALEN-NG-vector backbone	+	:* repeating mutagenesis-PCR on vector backbone psb1c3
-	• to do so a extension-PCR on the parts was done	+	:* extPCR of TAL-ORF (synthesis-product) in order to produce a n-terminal kind of adapter to paste recombinase
-	• CMV-Promotor was taken out of iGEM Distribution Kit 2012	+	:* 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
-	==  week 08/06/12 - 08/12/12 ==	+	:* transformation of mutated vector backbone
-	• mutagenesis-PCR of pcb1c3 vector backbone to eliminate restriction site for BsmBI	+	:* colony pcr to test provisory mammalian expression vector
-	• repeat of GGC to get MammoBrick	+	<br>
-		+	== 20/08 - 26/08 ==
-	• gel-run of mutagenesis-PCR of psb1c3	+
-		+
-		+
-	==  week 08/13/12 - 08/19/12 ==	+
-		+
-	• 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	+
-		+
-		+
-	== week 08/20/12 - 08/26/12 ==	+
-	• repeating mutagenesis pcr for vector backbone psb1c3 with another template	+	:* 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	+	:* finding a new method for successful extension pcr and then digest and ligation into the psb1cr backbone
		+	<br>
		+	== 27/08 - 02/09 ==
-	== week 08/27/12 - 09/02/12 ==	+	:* finding out that CMV promotor taken out of the registry distribution kit is not good at all
-	• finding out that CMV promotor taken out of the registry distribution kit is crap	+	:* trying to get another vector with cmv promotor and ordering new primer pair to amplify it
-	• 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
-	• 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
-	== week 09/03/12 - 09/09/12 ==	+	:* annealing of linker-part (was ordered as two single-strand primers)
-	• GGC-reaction in order to produce recombinase-linker-n-tal-orf construct	+	:* after ggc-reaction was done a pcr-amplification on the new assembled part was executed
-	• annealing of linker-part (was ordered as two single-strand primers)	+	:* gel-run shows a very weak band at 2,7 kb for hyperactive gin recombinase
-	• after ggc-reaction was done a pcr-amplification on the new assembled part was executed	+	:* GGC-reaction in order to clone direpeats for a complete TAL-Domain into provisory mammalian expression vector
-	• gel-run shows a very weak band at 2,7 kb for hyperactive gin recombinase	+	:* repeating the extextPCR on the recombinase-linker-n-tal-orf construct under different conditions after the first pcr reaction failed
-	• GGC-reaction in order to clone direpeats for a complete TAL-Domain into provisory mammalian expression vector	+	:* amplification of mutated psb1c3 vector backbone
-	• repeating the extextPCR on the recombinase-linker-n-tal-orf construct under different conditions after the first pcr reaction failed	+	:* trying to clone Transcription Factor into provisory mammalian expression vector
-	• amplification of mutated psb1c3 vector backbone	+	:* repeating of GGC reaction in order to produce MammoBrick
		+
		+	:* still working on our toolkit, doing the final steps: sent them off for sequencing
-	• trying to clone Transcription Factor into provisory mammalian expression vector	+	<br>
		+	== 10/09 - 16/09 ==
-	• repeating of GGC reaction in order to produce MammoBrick	+	:* 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
-	• still working on our toolkit, doing the final steps: sent them off for sequencing	+	:* 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
-	== week 09/10/12 - 09/16/12 ==
-	• 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>
	<br>		<br>
	<br>		<br>
		+	[[#top|Back to top]]

---

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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