Team:BIOTEC Dresden/Notebook3-2

4th October

The plates at 30°C were kept in the fridge.

5th October -The digestion from 2nd oct was repeated and the gel (1% TBE) was run again.

-7 colonies were inoculated in LB+Bsd+Spec from the plate containing Fosmid-SpecR+FRT-GFP-Bsd colonies.

-7 colonies were inoculated in LB+Kan+Zeo from the plate containing pTetFlp-KanR+F3-Zeo-F3-RFP colonies.

-2 plates were streaked with these colonies (Back up)

--> Fosmid-SpecR+FRT-GFP-Bsd (@ 37° C)

--> pTetFlp-KanR+F3-Zeo-F3-RFP (@ 30° C)

6th October

pTetFlp-KanR+F3-ZeoR-F3-RFP

-The 7 colonies did not grow because one used wrong concentration of Zeo (Zeo 50 instead of Zeo 15)

-There were several colonies growing on back-up plate.

-these 14 were inoculated into LS-LB+Kan30+Zeo15 and kept o/n @ 30°C

pRhaFlp-CmR-KanR

-18 minis were digested (7 from #18 to #24) that were previously digested on 2nd oct & 5th oct. And #1 to #11 of minis that were done before & not digested (on 23rd sep)

-400 ng/µl of these minis were digested using PvuII.

-Kept at 37°C for 2 hours.

Fosmid-SpecR-FRT-GFP-BsdR

-Nothing grew on plates from yesterday so Red/ET did not work.

-This has to be repeated!

so, o/n culture of GB20005 MN was kept @ 37°C.

7th October:

pTetFlp-KanR+F3-ZeoR-F3-RFP

The 14 minis were prepared, concentrations were checked and kept in fridge. These samples have to be digested and run on a gel, which will be done tomorrow.

pRhaFlp-CmR-KanR

The minis that were digested yesterday were run on a gel (1%).

1. 23 & #24 was run on another gel -no bands!

pRhaFlp-CmR - 4522, 1809, 593 bp

pRhaFlp-CmR-kanR - 2431, 2117, 1809, 593, 360 bp

1. 2, 5, 7 & 8 -clearly have a mixture!

has to be re-electroporated.

Fosmid-SpecR-FRT-GFP-BsdR

-FRT-GFPP-BsdR- digested using HincII

5 µl each was taken from the minis #3, 4,6 & #7 (Total 20 µl), 50 µl run on a gel and lower band was cut and purified. The remaining 50 µl was just stored in freezer (blue box).

-Red/ET was also done.

8th October:

F3-ZeoR-F3-RFP

digested using PvuII (samples from 1st oct- #1 and #2)

run on 1% gel and lower band (1500 bp) was cut and purified- kept in the freezer (4 tubes).

pTet-Flp-KanR+F3-ZeoR-F3-RFP

the 14 minis(7th oct)were digested PstI & run on a 1% gel.

1. 9, 12, & 14 were also re eletroporated, but for #9 and #14 the re-electroporation did not work, has to be repeated.

1. 12 re electroporated and plated over kan+zeo plate and #14 plated over zeo15.

9th October:

pTet-Flp-KanR+F3-ZeoR-F3-RFP

24 colonies were inoculated in LB+Kan+Zeo and kept o/n @ 30° C on a shaker (mini prep will be done on next day).

pRhaFlp-KanR-CmR

24 colonies were inoculated in LB+Cm+Kan and kept @ 37° C o/n ona shaker (mini prep will be done tomorrow)

Fosmid-SpecR+FRT-GFP-BsdR

4 plates (Bsd+Cm) were kept in the fridge, but then was growth on all plates (control too :( )

-waiting for the colonies to appear on the Bsd+Spec plates (They are still in the incubator @ 37° C)