Difference between revisions of "Team:Freiburg/Labjournals/Cloning/September"

Line 1: Line 1:
−
 
 
{{Freiburg/CSS}}
 
{{Freiburg/CSS}}
 
{{Freiburg/Menubar}}
 
{{Freiburg/Menubar}}
 
{{Freiburg/LabjournalCSS}}
 
{{Freiburg/LabjournalCSS}}
−
<html>
 
−
<div id="banner_labjournal">
 
−
</div>
 
−
</html>
 
 
{{Freiburg/wiki_content_start}}
 
{{Freiburg/wiki_content_start}}
 
<html>
 
<html>
−
<!-- Labjournal content goes in here -->
+
<style>
 +
.content_box {
 +
    background-color: #F9F9F9;
 +
    padding: 3em;
 +
    border: 3pt solid #5781BD;
 +
    border-radius: 30px;
 +
}
 +
</style>
 +
 
 +
<div class="content_box">
 
 
  
Line 387: Line 391:
 
</div>
 
</div>
 
<!-- EDIT14 SECTION "2015/09/06" [2822-] -->
 
<!-- EDIT14 SECTION "2015/09/06" [2822-] -->
 +
 +
</div>
  
 
</html>
 
</html>
−
<!-- Labjournal content ends here -->
 
 
{{Freiburg/wiki_content_end}}
 
{{Freiburg/wiki_content_end}}

Revision as of 15:31, 7 September 2015

""

2015/09/01

2015/09/02

Digest: pOP, pILS3 and K592009 with EcoRI (LS)

amountingredient
~5µlplasmid
5µlCut Smart
1µlEcoRI
up to 50µldH2O
  • incubation at 37°C, ~1h

PCR Purification of EcoRI digest (JN)

  • Qiagen kit
  • eluted in 20µl dH2O
concentration [ng/µl]
pOP 3.3
pILS3 23.
BBa_K592009 2.3

Digest of pOP, pILS3 and BBa_K592009 with PstI (JN)

ingredient amount [µl]
DNA16/17/18
PstI1
buffer 3.15
dH2O28/27/26
  • 37°C for 1h
  • analysis on 1% agarose gel

Gel-ex of BBa_I13504 (JN)

  • Qiagen kit
  • eluted in 20µl dH2O
  • concentration: 10.2ng/µl

Repetition of pOP Digest with EcoRI (JN)

ingredient amount [µl]
DNA5
EcoRI1
CutSmart5
dH2O39
  • 37°C for 1h

PCR Purification of pOP Digest

  • Qiagen kit
  • eluted in 20µl dH20
  • concentration: 3.7ng/µl

pOP digest with PstI

ingredient amount [µl]
DNA17
PstI1
buffer 3.15
dH2O27
  • 37°C for 1h
  • analysis on 1% agarose gel

Gel-ex of pOP (JN)

Ligation of pOP+BBa_I13504 (JN)

Trafo: Ligation of pOP BBA_I13504 in E.coli T10 (LS)

  • 10µl of ligation
  • transformation according to protocol

2015/09/03

Inoculation of pOP_BBa_I13504 (LS)

  • picked 3 clones
  • inoculation in LB-Amp (5ml)
  • incubation at 37°C

Inoculation of pOP_BBa_I13504 (JN)

  • 10 more clones were picked and inoculated o/n
  • each clone in 5ml of LB-Amp

2015/09/04

Mini-Prep of pOP_Anderson_GFP (JN)

  • all liquid cultures were fluorescent, only three were prepped, four more were pelleted in stored in the freezer as backup
  • Qiagen kit
  • eluted in 30µl dH2O
concentration [ng/µl]
pOP_Anderson_GFP 4 21.1
pOP_Anderson_GFP 9 42.9
pOP_Anderson_GFP 11 37.2
  • sample 9 and 11 were sent in for sequencing

Inoculation of pOP (JN)

  • inoculation of 3 colonies from already performed re-trafo (LS)
  • 3 liquid cultures with 5ml LB-Amp medium each
  • inoculated o/n

2015/09/05

Miniprep: pOP (LS)

  • 15µl of pOP
  • Zymo kit
  • elution in 65µl: 81,0 ng/µl

Digest: pOP and K592009 with EcoRI (LS)

amountingredient
10µl/~4µlpOP/K592009(all that was left)
1µlEcoRI
5µlCut Smart
up to 50µldH20
  • incubation at 37°C for 1h

Clean-up of Digest (LS)

  • Zymo PCR clean-up kit
  • elution in 20µl:
  • pOP:21,3ng/µl
  • K592009:2,7ng/µl

Digest: pOP and K592009 from clean-up with PstI (LS)

amountingredient
18µlpOP/K592009
5µlbuffer 3.1
1µlPstI
36µl dH2O
  • incubation at 37°C, 1h
  • analysis on 1% agarose gel:

-small band visible for pOP -nothing for K592009

Inoculation: 3x 5ml LB-Cml wit K592009 (LS)

  • incubation at 37°C, o/n

2015/09/06

Mini-Prep of K592009 (JN)

  • Qiagen kit
  • eluted in 20µl dH2O
177.0 ng/µl
294.4 ng/µl
364.6 ng/µl

Digest of all three preps with EcoRI (JN)

amountingredient
5µlK592009
1µlEcoRI
5µlCut Smart
39µldH20
  • incubation at 37°C for 1h