"
Team:Goettingen/NoteBook w9
From 2013.igem.org
| Line 10: | Line 10: | ||
| + | <div class="tlob" id="tl_0729"> | ||
| + | <span class="date">29th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span> | ||
| + | <div class="cont"> | ||
| + | <p class="timeline-title">Inoculation of 8 Clones from each, Riboswitch, DarR and E0840 (were put in the fridge on friday, plated 23. and 24.7.),Hybridization of iGEM_63 and iGEM_64 to generate the Promoter(part3)-Operator construct</p> | ||
| + | <div class="timeline-cont"> | ||
| + | <p class="c20"><span class="c5">Inoculation of 8 Clones from each, Riboswitch, DarR and E0840 (were put in the fridge on friday, plated 23. and 24.7.)</span></p><p class="c20"><span class="c5"> </span><span>-></span><span>E0840 heavily contaminated with something that looks like fungus. Plates discarded.</span></p><p class="c20"><span>->This leaves DarR and the Riboswitch for inoculation in 4ml Cam cultures for a miniprep tomorrow.</span></p><p class="c20"><span>-></span><span>in addition, a masterplate was plated.</span></p><p class="c20 c4"><span></span></p><p class="c20"><span class="c5">Hybridization of iGEM_63 and iGEM_64 to generate the Promoter(part3)-Operator construct</span></p><p class="c20"><span>The primers were first diluted in HPLC-H2O according to the QC-sheet (100µM). Then a 1:20 dilution stock was pipetted. For this experiment, the 1:20 dilution stocks were used.</span></p><p class="c20"><span>Protocol:</span></p><p class="c20 c75"><span>10µl iGEM_63</span></p><p class="c20"><span>+ 10µl iGEM_64</span></p><p class="c20"><span> 20µl reaction </span></p><p class="c20"><span>->80°C for 10 minutes (heatblock)</span></p><p class="c20"><span>->after 10 minutes, turn the heatblock off and let the sample slowly cool down in it</span></p><p class="c20"><span> </span></p><p class="c2 c21"><span>-></span><span> </span><span>The ends of the oligos are “digested” with EcoRI and SpeI and phosphorylated. The product of this experiment can directly be ligated in front of the GFP-part!</span></p><p class="c20"><span>See the gel tomorrow</span></p><p class="c20 c4"><span></span></p><p class="c20"><span class="c5">Restriction digestion of Part 1-4 and Part 7</span></p><p class="c2 c21"><span>-</span><span> </span><span>Part 1-4 digested with SpeI FD</span></p><p class="c2 c21"><span>-</span><span> </span><span>Part 7 digested with EcoR1+SpeI FD + Phosphatase treatment (last 20 minutes of digestion)</span></p><p class="c2 c21"><span>-</span><span> </span><span>40 µl setups, standard assay.</span></p><p class="c20"><span>->for part 1-4, restriction with PstI (not FD, because empty) on 30.7.</span></p><p class="c20 c4"><span></span></p> | ||
| + | |||
| + | <div class="fbutton">Fold ↑</div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
Revision as of 16:41, 17 September 2013
Inoculation of 8 Clones from each, Riboswitch, DarR and E0840 (were put in the fridge on friday, plated 23. and 24.7.),Hybridization of iGEM_63 and iGEM_64 to generate the Promoter(part3)-Operator construct
Inoculation of 8 Clones from each, Riboswitch, DarR and E0840 (were put in the fridge on friday, plated 23. and 24.7.)
->E0840 heavily contaminated with something that looks like fungus. Plates discarded.
->This leaves DarR and the Riboswitch for inoculation in 4ml Cam cultures for a miniprep tomorrow.
->in addition, a masterplate was plated.
Hybridization of iGEM_63 and iGEM_64 to generate the Promoter(part3)-Operator construct
The primers were first diluted in HPLC-H2O according to the QC-sheet (100µM). Then a 1:20 dilution stock was pipetted. For this experiment, the 1:20 dilution stocks were used.
Protocol:
10µl iGEM_63
+ 10µl iGEM_64
20µl reaction
->80°C for 10 minutes (heatblock)
->after 10 minutes, turn the heatblock off and let the sample slowly cool down in it
-> The ends of the oligos are “digested” with EcoRI and SpeI and phosphorylated. The product of this experiment can directly be ligated in front of the GFP-part!
See the gel tomorrow
Restriction digestion of Part 1-4 and Part 7
- Part 1-4 digested with SpeI FD
- Part 7 digested with EcoR1+SpeI FD + Phosphatase treatment (last 20 minutes of digestion)
- 40 µl setups, standard assay.
->for part 1-4, restriction with PstI (not FD, because empty) on 30.7.
