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Contents 1 Lab Work - 26/08/09 1.1 Stochastic Switch team 1.1.1 Summary 1.1.1.1 Running the gel 1.2 Metal Sensing Team team 1.2.1 Introduction 1.2.2 Practical Outline 1.2.3 Procedure 1.2.3.1 Restriction enzyme digests of the midi-prep DNA 1.2.3.2 DNA gel electrophoresis of digests 1.2.4 Results 1.3 Promoter Library Sub-Project

Lab Work - 26/08/09

Stochastic Switch team

Summary

Today Lab started at 2pm, so we had the afternoon to do restriction digests for the remaining biobricks(other than C0178). We used the fast digest system, which takes 5 minutes in a 37 degree waterbath rather than 60 minutes. Below are the volumes required for a double digest using EcoRI and PstI:

• 6ul water
• 2ul 10X Fast digest buffer
• 1ul of fast digest EcoRI enzyme
• 1ul of fast digest PstI enzyme
• 10ul DNA (miniprepped)

Total volume: 20ul

These were left for 5-10 minutes at 37 degrees, then 9ul of the total volume was added to 1ul of loading buffer, and the samples were run on the gel we prepared yesterday.

Running the gel

We loaded the samples in the following order

Sample	Location	Length
LuxR promoter	2,3	55bp
LasR promoter	4,5	157bp
LuxI CDS	6,7	585bp
LasR CDS	8,9	723bp
LasI	10	609
HixC	11,12	26bp

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Today we also prepared some overnight cultures of DH5alpha cells (2X 3ml plain LB) for the Genomic DNA prep which will be carried out tomorrow.

Metal Sensing Team team

Introduction

Practical Outline

The following list consists of the tasks the Metal Sensing team hope to achieve by the end of the day:

• Carry out the restiction enzyme digests of the midi-prep plasmid DNA (pSB1A2 containing BioBrick BBa_J33206) and run them on the gel.
• Carry out the genomic DNA preparations.

Procedure

Restriction enzyme digests of the midi-prep DNA

DNA gel electrophoresis of digests

Results

Promoter Library Sub-Project

The aim of this sub-project is to create a library of sigA promoters which differ in strength and to characterise them.

The first step in this process is to digest the pGFP-rrnb plasmid with restriction enzymes EcoRI and NheI. The enzyme digests were prepared in 1 Eppendorf tube in the following way:

• 15ul of pGFP-rrnb plasmid DNA (from a midiprep)
• 3ul NheI (FastDigest)
• 3ul EcoRI (FastDigest)
• 4ul FastDigest buffer
• 15ul distilled water

This makes up a total volume of 40ul The mixture was made in such a way that the DNA, the distilled water and the FastDigest buffer was added first before the enzymes were added. The tube was given a spin pulse and placed in a water bath at 37C for 1 hour.

After this the mixture was placed in the -20C freezer for use tomorrow.

In the meantime, 0.8% agarose gel was prepared in the usual way and poured into the gel tray. The only difference between this gel and others previously poured is that some of the comb was taped so that the wells formed were larger. Once the gel had set, 1xTAE buffer was sprinkled onto the surface and the gel wrapped up in cling film. The gel was placed in the fridge also for use tomorrow.