Team:Bielefeld-Germany/Labjournal/week1

From 2012.igem.org

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** Since we did not know the efficient of our competent KRX we used two different ''E.coli'' volumes for the transformation, 50µL and 100µL. We gave 50µL 10% Gylcerol to the reaction tubes with 1µL of the vector DNA (Bacillus pumilus). After the transformation we plated them into ampicillin plates.
** Since we did not know the efficient of our competent KRX we used two different ''E.coli'' volumes for the transformation, 50µL and 100µL. We gave 50µL 10% Gylcerol to the reaction tubes with 1µL of the vector DNA (Bacillus pumilus). After the transformation we plated them into ampicillin plates.
* '''Team Bacterial Laccase''': PCR with the ''Xanthomonas campestris B100'' and ''E. coli BL21(DE3)'' genomic DNA.  
* '''Team Bacterial Laccase''': PCR with the ''Xanthomonas campestris B100'' and ''E. coli BL21(DE3)'' genomic DNA.  
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Revision as of 12:52, 22 August 2012

Labjournal

Week1 Week2 Week3 Week4 Week5 Week6 Week7 Week8 Week9 Week10 Week11 Week12 Week13 Week14 Week15 Week16 Week17 Week18


Week 1 (04/30 - 05/06/12)

  • Start of our WET LAB time.
  • Generating new competent E.coli KRX cells.
  • Cultiviation of Xanthomonas campestris B100 and E. coli BL21(DE3). The bacterial strains we got from a working group at our University. After cultivation we isolated the genomic DNA. The DNA was needed as template for PCRs to purify the wanted laccase ORFs.
  • Sending requests to working groups for different plasmids , which have already worked with laccases and described them in their papers. Unfortunately just one answer came back (thanks a lot to them).So we got a vector with the laccase-ORF CotA from Bacillus pumilus ATCC7061 from the Swiss Federal Laboratories for Materials Science and Technology, Laboratory for Biomaterials in Switzerland.

Thursday May 3th

  • Team Bacterial Laccase: After the vector has arrived, we transformed it into the competent E.coli KRX which we have already generated. The protocol we used was as followed:
    • The electroporation setup: U= 2,5kV C= 25 µF and R= 400 <math>\omega</math>
    • Since we did not know the efficient of our competent KRX we used two different E.coli volumes for the transformation, 50µL and 100µL. We gave 50µL 10% Gylcerol to the reaction tubes with 1µL of the vector DNA (Bacillus pumilus). After the transformation we plated them into ampicillin plates.
  • Team Bacterial Laccase: PCR with the Xanthomonas campestris B100 and E. coli BL21(DE3) genomic DNA.
    • PCR table
Material Volume
Buffer (10x Phusion) 10µL
Phusion Polymerase 0,5µL
dNTPs 1µL
Primer Mix 1µL
Template DNA 1µL
DMSO 1,5µL
Water 35µL
    • PCR program
Temperature Time
1) 98°C 30 sec
2) 98°C 15 sec
3) 62°C 45 sec
4) 72°C 1 min
5) 72°C 3 min
6) 12°C

Cycle between step 2 and 4 35 times.

weekly seminar:

  • Do we want to order strains of Trametes versicolor and Trametes villosa?
  • Gathering information about signal sequences in yeast
  • Decision to create a database, so that we can easily number and inscribe our lab results
  • Decision to arrange a summer school for pupils in their last year before the final exams
  • Discussion about how to meet a member of the german Bundestag (the german parliament)
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