Reporter: Week 10 July 17-23

From 2011.igem.org

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|GFP
|GFP
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|Vector using AgeI and PstI (buffer 1)
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|Vector using AgeI and PstI (buffer 1):
 +
|tev cleavage site
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|Ligation
|Ligation

Revision as of 17:29, 2 August 2011

Sunday, July 17

No colonies grew for tev cleavage site+GFP, and only one colony grew for cI cleavage site+GFP. This colony was amplified through colony PCR (extension time=1:20). The PCR products were run on an agarose gel, which yielded a band that corresponded to around 1000 bp, which was expected. The colony was grown up in culture overnight.

Monday, July 18

The tev mutagenesis and cI cleavage site+GFP plasmids were sent to the sequencing center. The sequencing results showed that the cI cleavage site+GFP worked, but the tev mutagenesis did not.

Since we have had trouble inserting the cleavage sites in front of the GFP gene, the GFP was inserted behind the cleavage sites.

Protocol Part Involved with Protocol
Insert PCR (extension time=1:15) GFP
Restriction Digest Insert using NgoMIV and PstI (buffer 1) GFP
Vector using AgeI and PstI (buffer 1): tev cleavage site
Ligation tev cleavage site+GFP
Transformation/Plating The above ligation was transformed into
Escherichia coli cells and plated on
kanamycin resistant plates.

The promoter and RBS construct (J23100+B0034) was placed in front of GFP+tev cleavage site, XylE+small linker and XylE+Imp linker following the procedure performed on Thursday, July 7.

The GusA and LacZα genes were cloned into K3 using the 10AA linker+K3 as the vector.

Tuesday, July 19

The promoter and RBS colonies that started growing on Monday, July 18 were amplified through colony PCR (extension time=1:15). The PCR products were run on an agarose gel, which yielded bands that corresponded to the correct number of base pairs. These colonies were grown up in culture. The J23100+B0024+XylE+10AA linker and J23100+B0024+GFP+cI cleavage site cultures were frozen as stocks.

The GusA and LacZα genes were cloned into K3 following the same procedure performed on Monday, July 18.

The linkers were placed in front of XylE.

Protocol Part Involved with Protocol
Restriction Digest Insert using NgoMIV and PstI (buffer 1): XylE
Vector using AgeI and PstI (buffer 1): 10 AA linker
Imp linker
small linker
Ligation 10AA linker+XylE
Imp linker+XylE
small linker+XylE
Transformation/Plating The above ligations were transformed into
Escherichia coli cells and plated on
kanamycin resistant plates.

Results: The 10 AA liker+XylE worked, but the Imp linker+XylE and small liker+XylE must be repeated.



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