Team:BIOTEC Dresden/Notebook3-3

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)