"
Team:Bielefeld-Germany/Labjournal/July
From 2013.igem.org
(Difference between revisions)
| Line 163: | Line 163: | ||
<div class="cyt_cleanup1"> | <div class="cyt_cleanup1"> | ||
<blockquote> | <blockquote> | ||
| - | Gelextraction and clean up with the AnalytikJena GelExtraction-Kit | + | <u>Gelextraction and clean up with the AnalytikJena GelExtraction-Kit</u> |
<p>-Elution buffer was preheated to 50°C </p> | <p>-Elution buffer was preheated to 50°C </p> | ||
<p>-We eluted each column twice, with 20 ul each and combined it afterwards</p> | <p>-We eluted each column twice, with 20 ul each and combined it afterwards</p> | ||
| - | <br>NanoDrop | + | <br><u>Measurement of nucleid acid concentration via NanoDrop </u> |
<p>-Fragment1: 4-2607-304: 10.5 ng/ul</p> | <p>-Fragment1: 4-2607-304: 10.5 ng/ul</p> | ||
<p>-Fragment2: 4-2707-003: 44.6 ng/ul</p> | <p>-Fragment2: 4-2707-003: 44.6 ng/ul</p> | ||
Revision as of 20:47, 2 September 2013
July
Milestones
1.Week
MFC
Mediators
Cytochromes
The mtrCAB fragment has two illegal PstI restriction sites at x xbp and yy bp, so we had to design new primers to remove them. We replaced one base in each restriction site, without affecting the coding triplett, by respective primer overlaps . We ended up with three different fragments, which will be ligated back together via Gibson Assembly.
mtrCAB_Frag1_rev:
mtrCAB_Frag2_fwd:
mtrCAB_Frag2_rev:
mtrCAB_Frag3_fwd:
Biosafety
Porines
2.Week
MFC
Mediators
Cytochromes
Biosafety
Porines
3.Week
MFC
Mediators
Cytochromes
Biosafety
Porines
4.Week
MFC
Mediators
Cytochromes
Size:1800 bp
Program: PhusionPCR
Gradient: 54°C - 71°C over 8 steps
Primer: mtrC_fwd & mtr_Frag1_rev
Template: S. oneidensis PCR Template from genomic DNA
Notes: Annealing temperature has no significant impact on the PCR
Size:330 bp
Program: PhusionPCR
Primer: mtr_Frag2_fwd & mtr_Frag2_rev
Template: S. oneidensis PCR Template from genomic DNA
Notes:
Size:3000 bp
Program: PhusionPCR <
Gradient: 54°C - 71°C over 8 steps
Primer: mtr_Frag3_fwd & mtrB_rev
Template: S. oneidensis PCR Template from genomic DNA
Notes: Annealing temperature has no significant impact on the PCR
Gelextraction and clean up with the AnalytikJena GelExtraction-Kit-Elution buffer was preheated to 50°C
-We eluted each column twice, with 20 ul each and combined it afterwards
Measurement of nucleid acid concentration via NanoDrop-Fragment1: 4-2607-304: 10.5 ng/ul
-Fragment2: 4-2707-003: 44.6 ng/ul
-Fragment2: 4-2707-004: 13.5 ng/ul
-Fragment3: 4-2607-301: 11.1 ng/ul
-Fragment3: 4-2607-302: 8.9 ng/ul
-Fragment3: 4-2607-303: 9.2 ng/ul
