Transfection procedures

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Introduction
We will express recombinant proteins in COS or HEK293 Cell s Ð see table below for further details. COS (HEK293) cells will be provided at 50-70% confluency. We will use three different transfection procedures/reagents, DEAE dextran, calcium phosphate and Effectene™.

Aims
You will work individually, to perform your transfections Ð two people will work at the same time in the tissue culture facilities in the Wolfson building.

Caution
Class II GMOs Ð This practical involves the use of category II genetically modified organism -please follow instructions by demonstrators for safe handling and disposal.

Timescale
Cell s must be split to an appropriate density, one or more days prior to transfection. Different transfection procedures take differing lengths of time - methodical planning is the key to success.

Transfection strategy

Effectene Transfection
Materials required
Condensation buffer (Buffer EC) pEGFP-N1
Effectene Transfection Reagent pP2X2-EGFP
Enhancer pGL3-BAS & pGL3-CON
1x PBS pcDNA3.1+