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Team:Bielefeld-Germany/Labjournal/week5
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* '''Team bacterial laccase''': Restriction of ''E. coli'' CueO, ''Xanthomonas campestris'' CopA and the ''B. pumilus'' laccase CotA and the pSB1C3 + RFP plasmid with the same restriction enzymes, ligation of the restricted products and transformation. | * '''Team bacterial laccase''': Restriction of ''E. coli'' CueO, ''Xanthomonas campestris'' CopA and the ''B. pumilus'' laccase CotA and the pSB1C3 + RFP plasmid with the same restriction enzymes, ligation of the restricted products and transformation. | ||
| + | ** Since we have less CopA and tth-laccase DNA, we set an PCR from the remaining PCR approach. For the reaction look [https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week1 PCR table] | ||
===Tuesday May 29th=== | ===Tuesday May 29th=== | ||
Revision as of 08:56, 5 September 2012
Contents |
Labjournal
Week1 Week2 Week3 Week4 Week5 Week6 Week7 Week8 Week9 Week10 Week11 Week12 Week13 Week14 Week15 Week16 Week17 Week18 Week19
Week 5 (05/28 - 06/03/12)
Monday May 28th
- Team bacterial laccase: Restriction of E. coli CueO, Xanthomonas campestris CopA and the B. pumilus laccase CotA and the pSB1C3 + RFP plasmid with the same restriction enzymes, ligation of the restricted products and transformation.
- Since we have less CopA and tth-laccase DNA, we set an PCR from the remaining PCR approach. For the reaction look PCR table
Tuesday May 29th
- Team Activity Tests: Our ordered laccase and ABTS arrived. We couldn't wait to start, so we set up some stocks and created an ultimate plan of how to get to know our laccase better. We found out that natrium acetate buffer (100 mM / pH 5) would give an optimal environment to our enzyme. We decided to check the activity via a photometer. The one we may use here at the Cebitec is a Tecan Microplate reader. Check "protocols" for further information. If oxidized by laccase ABTS can me measured at 420 nm. After some trial and error we found out that a concentration of 0,1 U laccase and 0,1 µl ABTS in each well is perfect for visualizing the process. We added buffer to fill each well to 200 µl.
- Team Student Academy: E. coli Mach1 with pMTE cp46 His received from the working group “Fermentation Engineering”, University Bielefeld. Plasmid contains genes for GFP and kanamycine resistance. We plated it and made a liquid culture at 30°C. Result: There was an intense fluorescence. We made a glycerol stock and a plasmid isolation.
Wednesday May 30th
Thursday May 31st
- Team bacterial laccase: The PCRs from the 28th May has been purified with the Fermentas PCR Clean Up kit.
