Team:Goettingen/NoteBook w8

Innoculate ON culture for plasmid mini-prep

Part7 C1

RBS-GFP-Term construct C1,C4,C5

Test restriction of plasmid RBS-GFP-Term:

C2 as control, C1,C4,C5

 --->the Part6 already has the RBS and Term.

 [that simply makes the work above meaningless]

For the construct of  RBS-DarR ORF-Term construct (expression construct)

DarR ORF into Part8(PBS plasmid)

     suffix insertion { cut part8 with SpeI and PstI, and the DarR ORF with XbalI and PstI }

RBS-DarR ORF into part7(Term plasmid)

     Prefix insertion {cut part7 with EcoRI and XbaI, the RBS-DarR ORF with EcoRI and SpeI)

The first round of part8 digestion with SpeI: to have linearized structure for DarR ORF into Part8

        Fast SpeI: 4ul

        FD buffer : 4ul

        part8 DNA: 1.5ug

        Water: fill up to 40ul

Digestion:1.5 hour, not fully digested

        Stored at -20 overnight

          tbc

Restriction digestion of part 7

-          template from stock

-          restriction with EcoRI and PstI FD, gel extraction to remove terminator out of the construct

-          Nanodrop meassurment 5,3 ng/µl

-          Inocculation from cryostock. 5h, only 2h on the shaker (stupid me). Yield after miniprep way to low (10x 50 µl with 1-3 ng/µl)

-          →redone on 23.7 overnight inocculation

(pic to be added)