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Contents 1 Lab Work - 02/09/09 1.1 Promoter Library Sub-Project 1.1.1 Introduction 1.1.2 Preparing the gel and the DNA gel electrophoresis

Lab Work - 02/09/09

Promoter Library Sub-Project

Introduction

In yesterday's session, PCR amplifications for the three types of sigA promoter variants was carried out. The amplifications of these three types of promoter variants will give rise to a library of different sigA promoters, hopefully of differing affinities.

In today's session the PCR products will be run on the gel and analysed. If there are signs that the PCR reactions have worked successfully then the PCR products will be purified. If it is apparent that the PCR reactions haven't worked at all then another attempt at PCR shall be made.

Preparing the gel and the DNA gel electrophoresis

The fragments we are expecting to be seen in the gel are roughly 100bp long therefore a fine gel is needed to capture these PCR products. Instead of making up 0.8% agarose gel, 1.5% agarose gel was made up and melted in the way the protocol dictates.

With the gel now set in the tray 6ul (5ul of PCR product and 1ul of loading buffer) of each PCR product was placed into the wells of the gel. The electrophoresis machine was switched on to 90mv and the process allowed to run for 30 minutes.

The resulting gel photograph can be seen here:

At first glance it seems that the PCRs have completely failed but on closer inspection there are little clouds of flourescing material just below the second row of dark bands in the middle of the lanes (caused by the colour bands). These could well be PCR products; another attempt at gel electrophoresis would clarify this. It is possible that the DNA gel electrophoresis has been left to run too long and additionally the voltage has been set too high.