Team:Newcastle/Labwork/25 August 2009

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Contents 1 Stochastic Switch team 1.1 Summary 1.2 Restriction digest 1.2.1 Running the gel 1.3 Preparing the DNA ladder 2 Metal Sensing Team team 2.1 Introduction

Stochastic Switch team

Summary

Today we decided we should redo the minipreps from last weeks biobricks using the overnight cultures we set up yesterday. For the miniprep procedure we followed the extra guidelines that Chris advised us about. The final rehydrated plasmid minipreps were put into the -20 freezer in the 'iGEM plasmid minipreps' box. We also did a restriction digest of the two miniprep samples for biobrick C0178 and ran this on a 0.8% agarose gel. This went well, both samples having the fragment sizes expected. Tomorrow we will be carrying out another restriction digest of todays minipreps, however to save time tomorrow, we poured a gel and put it into the fridge ready to load the samples tomorrow. In order to make sure the prepared gel did not dry out, a small amount of 1% TAE was poured onto the gel, which was wrapped in cling film, clearly labelled and put into the fridge.

Restriction digest

Today we did restriction digest for a miniprep prepared for BBa_C0178. We prepared DNa ladder to load to the gels. At the end of the day, we prepared our gel to run tomorrow.We followed the restriction digest protocol for our two miniprep prepared for BBa_C0178 and used the followings to prepare the solutions.

• H20, 6.5 ul
• 10x Buffer, 2ul
• DNA from minipreps, 10ul
• EcoRI, 0.5 ul
• PstI, 0.5 ul
• BSA, 0.5 ul (It was diluted 100 times)

We gave a quick pulse to mix everything in the tubes before we put them into 37C incubator for an hour.

Running the gel

Metal container team had already prepared the gel for us. We placed the gel tray into the gel electrophoresis device and top it up with 1xTAE buffer. 9ul of DNA from each tube is combined with 1ul of buffer and loaded into the wells. DNA ladder was loaded into the 1st and the 4th wells, DNAs were loaded into the 2rd and the 3rd wells. Restriction digest should have cut the plasmid(pSB1A2) into two pieces, plasmid and the the biobrick, from EcoRI and PstI sites. pSB121A2 is 2079bp and BBa_C0178 is 609bp long. As it can be seen from the results below, they match with the HindIII markers.

Preparing the DNA ladder

We diluted the Lambda DNA/HindIII marker 10 times with the loading dye as below and placed the tube containing the mix into the fridge

• 360ul loading dye
• 40ul Lambda DNA/HindIII

Metal Sensing Team team

Introduction

Yesterday's lab session saw the DNA gel electrophoresis of the mini-preps of the BioBrick BBa_J33206 and it's plasmid pSB1A2; both cut and uncut with restriction enzymes. Yesterday's lab session also saw a midi-prep of the BBa_J33206 BioBrick which was then transferred to the freezer for storage.

However not everything is as straightforward as it seems; the bands exhibited when restriction enzyme-treated mini-prep samples were run on the gel yesterday weren't what was expected. Whilst the backbone fragment looked realistic, the small insert fragment looked significantly smaller than it should have done. As suggested yesterday this result could be due to the incompleted work of the FastDigest restriction enzymes (which had been newly ordered). This incompleted work could be affected by the time given for the enzymes to work or may be due to it's efficiency.

Yesterday's lab session also involved Team Newcastle's first attempt at carrying out a PCR reaction. To prove that the plasmid pGFP-rrnB had truely integrated into the amyE gene in the Bacillus subtilis genome (in those B. subtilis which had been transformed), the Metal Sensor team carried out PCR reactions using primers which correspond to regions within the amyE gene in the Bacillus genome. Any PCR products that arise from the different samples we used can determine whether the plasmid has truely integrated.

In any case today's work involves removing the ethanol from the DNA and concentrating it (as observed in the midi-prep protocol). We also hope to make some agarose gel up and run the digests of the midi-prep samples on it through DNA gel electrophoresis. In addition to these tasks, we hope to prepare the PCR 'results' and run them on the gel in DNA gel electrophoresis; once analysis has been made and the verdict given we can decide on where we go from here.