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Introductory Lab Sessions

(23rd July - 24th July 2009)

The fourth week initial planning, 23rd July - 24th July

• Thursday
• Friday

Lab session: 23rd July

Introduction

In the last session of last week’s set of practicals, we had attempted to conduct a midi-prep of the plasmids containing the RFP and the GFP genes (derived from E.coli DH5alpha cultures 2 and 3 respectively). However we were unsuccessful in loading the two prepared plasmids into the gel in our electrophoresis attempt. When inserted into the wells the DNA immediately floated and dispersed within the running buffer of the gel electrophoresis apparatus.

The reason for this anomalous result was that the samples still contained a high concentration of ethanol, which should have been completely washed away prior to loading into the wells. Washing solution 2 contained ethanol as an ingredient. After analysis of the situation, a few conclusions were drawn:

• For the washing process 15ml falcon tubes should have been used instead of 50ml falcon tubes.
• The centrifugation step should have been allowed to run for longer to ensure all of the wash solution had passed through the buffer column.

However all was not lost; the plasmid DNA was still there and in usable form. The challenge was how we could extract it successfully.

The answer to our problem is ethanol precipitation. By precipitating the ethanol from our DNA sample, we could then process the plasmid DNA and run on the gel as usual.

In the meantime we also need to grow up cultures containing the plasmid backbone pMUTIN4, which will allow us in the near future to transfer our chosen BioBricks from the E.coli cells we are working with into Bacillus subtilis.

Another task which we have to complete is to prepare the BioBricks which are needed for our system and are found in the Spring Distributions.