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Team:Bielefeld-Germany/Labjournal/week1
From 2012.igem.org
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)
