Team:Newcastle/22 June 2010

Aims

The aim of today's Lab practice was to extract the plasmids for GFP and RFP, digest the 2 plasmids and extract the 2 inserts and 1 of the backbones (vector)from an agarose gel. From this a ligation was set up.

Materials and Protocol

QIagen Miniprep Kit Using a Microcentrifuge for Plasmid Extraction

Please refer to: Qiagen Minipreps for materials required and protocol.

Digest

Please refer to: Restriction digests for materials required and protocol.

Gel extraction

Please refer to: Gel extraction for materials required and protocol.

Agarose Gel Electrophoresis

Please refer to: Gel electrophoresis for materials required and protocol.

Dr Wendy demostrating how to use gel electrophoresis machine

Gel tank

Deena pouring hot molten gel

Cut gel out

Putting the gel on the geldoc

Preparing to cut the gel

Gel illuminated by the UV light which is emitted by the geldoc

We cut the inserts which were about 900bp and we use the backbone from the plasmid consisting rfp.

QIAquick Gel extraction kit

Phil cutting the gel

Transferring the cut gel part into a 2 ml microfuge tube

Set up ligation

Transformation protocol

Please refer to: Transformation of E. coli for materials required and protocol.

Nanodrop Protocol

Please refer to: Nanodrop Spectrophotometer for materials required and protocol.

A typical curve to be achieved from a nanodrop spectrophotmeter

Phil analyzing a sample on the nanodrop machine

Analyzed data on the screen

Outcome