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From 2011.igem.org

Contents 1 Sunday, June 26 1.1 Insert tev Protease into K3 Vector, Take 7 Day 1 2 Monday, June 27 2.1 Insert tev Protease into K3 Vector, Take 8 Day 1 2.2 Cloning of XylE and GFP 2.3 Catechol Assay, Day 2

Sunday, June 26

Insert tev Protease into K3 Vector, Take 7 Day 1

     The plates containing the assembly from 6/25 grew colonies that only contained RFP, none of which contained the tev protease. Thus, this assembly must be done again.

     The purified tev protease PCR product from 6/4 and the K3 miniprep from 6/25 were digested with XbaI and PstI in buffer 3. The digests were ligated together, then the ligation was transformed into competent Escherichia coli cells and plated onto kanamycin resistant plates.

Monday, June 27

Insert tev Protease into K3 Vector, Take 8 Day 1

     The colonies that grew overnight all contained RFP, meaning that the insertion of tev protease into K3 from 6/26 did not work. As a result, we performed the insertion again. We started with the tev protease (K316017) plasmid from the 6/8 miniprep and the K3 vector miniprep.

Protocol	Part Involved in Protocol
Insert PCR	K316017
Restriction Digest	Insert using XbaI and PstI:	K316017
	Vector using XbaI and PstI:	K3
Ligation	K316017+K3
Transformation/Plating	The ligation above was transformed into Escherichia coli cells then plated on a kanamycin resistant plate.

Cloning of XylE and GFP

     The purified PCR products of the mutated XylE and GFP were digested with XbaI and AgeI. The Imperial Linker + K3 served as the vector and was digested with XbaI and AgeI. The two digests were ligated together, transformed into Escherichia coli cells and plated onto kanamycin resistant plates.

Catechol Assay, Day 2

Two 5 ml M9 cultures of K316009 stock were grown up at 37°C with shaking.

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