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Team:Goettingen/NoteBook w9
From 2013.igem.org
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Revision as of 16:43, 17 September 2013
29th
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.
