Team:Goettingen/week6-3

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#3 Chemoreceptor Library - 6th Week

Preparation of pUC18_TAR_QC

Experiment:
1% agarose gel was performed to analyze DpnI digest. Gel extraction was performed as described before with PeqGOLD Gel extraction kit (Peqlab). Samples were stored at -20 °C.

Transforamtion of DH10B with pUC18_TAR_QC

Preparation of Miniprep cultures

V06_07_1 Preparation of chemocompetent cells of Δtar

Experiment:
Chemocompetent cells were prepared as described in this protocol. ON cultures from 06.06. were used.

V06_07_2 Miniprep of pUC18_TAR_QC

Experiment:
Minipreps were done with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual. A test digestion was performed using XbaI and the expected fragment of 4342 bp was observed on the corresponding gel. Minipreps were stored at -20 °C.

V06_08_1 Preparative double digest of pUC18_TAR_QC

Experiment:
Digest was performd with XbaI and PstI (according to BioBrick standard) according to protocol. A gelelectrophoresis of the digest was performed which showed the expected bands at 2662 bp and 1680 bp. The insert TAR_QC (1680 bp) was cut from the gel and a gel extraction was performed as described before. Stored at -20 °C.

V06_08_2 Sequencing of pUC18_TAR_QC

Experiment:
Sequencing reactions were done by Prof. Daniels Laboratory (University of Göttingen), Göttingen Genomics Lab and set up according to their protocol. Primers used: pUC18_M13 for, pUC18_M13 rev, pUC18_TAR_Seqfor01, pUC18_TAR_Seqfor02. Dilutions 100 μM, 10 μM, 5 pmol.