Revision as of 12:11, 22 August 2012

Labjournal

Primerdesign for isolating a laccase from Arababidopsis thaliana cDNA. Since we want to express the laccase in E.coli we designed the primers like before for the bacterial laccases with T7 promotor and HIS-tag.
Team bacterial laccase: The bacteria S. griseus and S. lavendulae has been delivered so we can start with PCRs. We set the first PCR with them as followed:
- PCR table

Cycle between step 2 and 4 35 times.

Team Activity Tests: After testing the T. versicolor laccase under conditions that are optimal (pH 5, 25°C ) according to the literature we now started further characterization under different pHs. We analyzed the laccases behavior when working in 100 mM natrium acetate buffer at pH 1, 3, 7 an 9. Result: We agree with the literature that pH 5 and also pH 6 seem to make the laccase happy. Since not all waste waters (especially those here in Germany) are not as warm as 25°C we now wonder what our laccase might do when exposed to low temperature such as 4°C. Stay tuned.

Team Wiki: Yay for Team Wiki´s first entry. Our first steps with the iGEM Bielefeld 2012 Wiki contain thinking about contents, layouts, programming and responsibilities. Our first rules are:
- we are programming static pages in HTML and all the other pages (those that will be updated by all team members) in wiki code.
- we created all pages and will fill them up with some nice and beautiful content constantly from now on.
- Our temporary banner contains our outstanding logo and a DNA but we will set up a new layout soon.

@@ Line 28: / Line 28: @@
 * Primerdesign for isolating a laccase from ''Arababidopsis thaliana'' cDNA. Since we want to express the laccase in E.coli we designed the primers like before for the bacterial laccases with T7 promotor and HIS-tag.
-* '''Team bacterial laccase''': The bacteria ''S. griseus'' and ''S. lavendulae'' has been delivered so we can start with PCRs.
+* '''Team bacterial laccase''': The bacteria ''S. griseus'' and ''S. lavendulae'' has been delivered so we can start with PCRs. We set the first PCR with them as followed:
+** '''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
+|-
+| Water || 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.
 ===Tuesday June 5th===