Team:Bielefeld-Germany/Labjournal/week1

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(→Labjournal)
(→Week 1 (04/30 - 05/06/12))
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* 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.  
* 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.   
* 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'''
 +
{| class="wikitable"
 +
|-
 +
! Material !! Volume
 +
|-
 +
| Buffer (10x Phusion) || 10µL
 +
|-
 +
| Phusion Polymerase || 0,5µL
 +
|-
 +
| dNTPs || 1µL
 +
|-
 +
| Primer Mix || 1µL
 +
|-
 +
| Template DNA || 1µL
 +
|-
 +
| DMSO || 1,5µL
 +
|-
 +
| Watter || 35µL
 +
|-
 +
|}
 +
''' PCR program'''
 +
{| class="wikitable"
 +
|-
 +
! 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:
weekly seminar:

Revision as of 11:13, 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
Watter 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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