Team:Goettingen/week5-3

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#3 Chemoreceptor Library - 5th Week

Insertion of tar into pUC18

Experiment:
Double digest of tar and pUC18 using EcoRI, PstI and Orange buffer (ThermoScientific) according to protocol. Gel extraction and purification of pUC18 and tar using PeqGOLD Gelextraction Kit (Peqlab). Ligation of tar into pUC18 using T4 DNA Ligase (Thermo Scientific).

Transformation of DH10B with pUC18_TAR construct

Experiment:
Transformation was carried out according to protocol. Transformed DH10B were plated on LB-Amp plates (see here for recipe). Transformation was successful.

Preparation of pUC18_TAR

Experiment:
Inocculation of overnight cultures of 10 supposedly positive clones in liquid LB-Amp (recipe).

Changing the XbaI site in the tar sequence via QuikChange

Experiment:
DNA was prepped using PeqGOLD MiniPrep Kit (Peqlab). A DNA test digest with EcoRI and XbaI was performed according to protocol and analyzed on a 1% agarose gel. Test digest showed 8 out of 10 clones contained the construct pUC18_TAR. QuikChange PCR was performed using designed primers and Pfu Turbo polymerase (see QuikChange protocol here). Resulting construct was labeled pUC18_TAR_QC.

Changing the XbaI site in the tar sequence via QuikChange

Experiment:
DpnI digest of QuikChange PCR product to get rid of the parent plasmid.