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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.

Practical Outline

1. Precipitate the ethanol from our DNA plasmids (containing the RFP and GFP genes) and analyse them through DNA gel electrophoresis.
2. Grow up some DH5alpha E.coli cells containing the pMUTIN4 plasmid vector
3. Hydrate and prepare BioBricks present in Spring Distributions.

Ethanol Precipitation

We used the ethanol precipitation protocol and carried out these steps:

• Added 0.2ml of sodium acetate (a tenth of the volume of DNA) to 2mls of DNA from both GFP and RFP samples.

• To these mixtures, 6.6ml of 100% ethanol was added.

• The 8.8ml of GFP DNA + sodium acetate + ethanol solution was then divided into 8 Eppendorf tubes – each Eppendorf tube containing 1ml of solution.

• In the same way, the 8.8ml of RFP DNA + sodium acetate + ethanol solution was then divided into 8 Eppendorf tubes – each Eppendorf tube containing 1ml of solution.

• Both sets of Eppendorf tubes were then stored in the fridge for further work tomorrow.

Preparing cultures containing pMUTIN4

For this exercise 2 x 500ml flasks were used. Into each flask 100ml of LB solution was added (under aseptic conditions) and also to each flask 0.5ml of ampicillin was added (ampicillin resistance is conferred by the pMUTIN4 plasmid). A sample was then taken from an agar plate containing E.coli possessing pMUTIN4 and was added to each of the two flasks. The 500ml flasks were then left in the 37ºC shaking incubator overnight.