Team:BIOTEC Dresden/Notebook3-3
From 2009.igem.org
12th October:
Miniprep concentrations were checked for
pRha -CmR-KanR
from 10th Oct.
p Tet-Flp-KanR + F3
pTet-Flp-KanR+F3-ZeoR-F3-RFP digested with PstI and got all nice bands...and we got our first final construct!! :D
PRhaFlp-CmR-KanR digested with PvuII
Fosmid-SpecR+FRT-GFP-BsdR
-22 colonies were inoculated in LB+Cm+Bsd for min-prep, which will be done tomorrow.
An o/n culture of GB05-DIR was set up and kept @ 30°C (incubator).
Silver peptide stuff:
Primers arrived- Ag-Kan fwd
Ag-Kan Rev
PCR reaction was prepared (PCR mix) and performed.
13th October:
pTet-Flp-KanR+F3-ZeoR-F3-RFP
10 µl each from #4, #5 and #9 minis from yesterday were digested using BspEI (pRhaFlp-CmRKanR)
F3-ZeoR-F3-RFP
digested with XhoI (10 µl from #8 and 20µl from #9) from plasmid database.
the samples run on 1% TBE and following bands were cut from the gel:
F3-Zeo-F3-RFP- 1400 bp
pRha-Flp-CmR-KanR - 6400 bp
the digested parts were purified from the gel and concentration werechecked.
8 tubes were set up containing 1.4 ml of LB for Red/ET recombination ....and another mistake was made, added insert in wrong samples
pTet-Flp-KanR+F3-ZeoR-F3
4 minis were retransformed (#1, 17, 19 and 20), #19 and #20 are in the main plasmid database.
-plated on Kan30 plate, kept @30° C o/n.
Fosmid-SpecR+FRT-GFP-BsdR
22 minis were done.
Concentration of minis were checked and digested with EcoRI.
14th October:
pRha-Flp-CmR-KanR
- 8 tubes with 1.4 ml of LB were inoculated with 40 µl of the o/n GB05-DIR cells for Red/ET recombination.
and Red/ET is done., plates (LB+Cm+Kan+Zeo) kept in incubator at 30° C o/n.
Fosmid-SpecR+FRT-GFP-BsdR
The samples digested yesterday were run on a gel (1% TBE)