Team:Newcastle/Labwork/27 August 2009

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Stochastic Switch team

Summary

Today Goksel did a restriction digest of our plasmid backbone (pSB1AT3) cutting out the mCherry coding sequence. This will prepare the backbone for the PCR products of ara, sac and sspb, which we intend to build as a construct within this plasmid. The sample was run on a gel and, using the transilluminator, the correct band was cut out of the gel. This can be purified tomorrow.

Today we also did a Genomic DNA prep for E.coli. We need this for our PCR reaction in order to get the sspb degradation controller protein. We used the gram negative protocol provided in the kit. Yesterday we set up 2 ON cultures for DH5alpha E.coli:

• We spun down the cultures to pellet them, and aspirated the media.
• The pellet was resuspended in lysis solution T
• For RNA free DNA we added RNAse and incubated for 20 minutes.
• Proteinase K was then added and the solution incubated in the waterbath for 30 minutes at 55C
• Then the Lysis solution C was added and was incubated for a further 10 minutes at 55C
• During this time the column was prepared to the binding column.
• Ethanol was added to our solution and the bind, wash and elution steps were carried out as the protocol suggested.

Sigma Genelute Bacterial Genomic DNA kit protocol

We will run our results on a gel tomorrow as well as analysing them on the nanadrop in order to determine DNA concentration and quality.

Restriction digest

Before we start using pSB1AT3+mCherry from the midipreps we diluted the tube with 100ul of water. We then followed the steps to cut the plasmid from EcoRI and SpeI sites to remove the biobrick. The plasmid backbone will be used for our parts later on.

• 15ul of water
• 4ul of fast digest buffer
• 15ul of DNA(pSB1AT3)
• 3ul of fast digest EcoRI
• 3ul of fast digest SpeI

After mixing everything we added 50ul of the loading buffer to make the final volume 50ul.

Running the gel

We prepared the gel with large wells to be able t load 50ul. We used 10ul of DNA ladder.

We then cut the gel fragment containing the plasmid backbone.

The fragment was placed in an ependorf tube and weighed as 184mg. It was then placed into the fridge for tomorrow.

Promoter Library Sub-Project

Introduction

Yesterday's lab session saw pGFP-rrnB DNA being cut with restriction enzymes EcoRI and NheI which is now stored in the -20C freezer. For today's lab session, we hope to run the enzyme digested pGFP-rrnB products on agarose gel (also made yesterday) and use this analytical procedure to isolate the 7995Kb plasmid fragment. Once the fragment has been determined by exposure to ultra-violet light, we hope to cut it out of the gel and extract the DNA from the agarose using a gel extraction kit.

Extracting the 8Kb fragment

Prof. Wipat updating lab-book before carrying out the gel extraction process

40ul of the DNA + restriction enzyme solution was run on the gel for about 10-20 minutes under 70mv before the electrophoresis was stopped. The gel was then taken to the dark room (where the gel analysis equipment is kept) and under UV light, the fluorescing band found at the top of the well was cut out of the gel. The fragment was then placed in an Eppendorf tube for the gel extraction process. Although the heavier band was taken for processing, it has to be said that we were unconvinced that the digestions had worked properly; the bands we had expected to see did not emerge and the probable reason for this the time these enzymes had to operate. The FastDigest enzymes had been used and despite the protocol suggesting these enzymes take 5 minutes to digest, the bands seemed to show otherwise.

Meanwhile, the 8kb fragment of DNA was prepped using the gel extraction kit; the end solution was 50ul eluted plasmid DNA which is now stored in the -20C freezer.

Another attempt at digesting pGFP-rrnB with EcoRI and NheI

Once this had been accomplished, another attempt to digest the pGFP-rrnB plasmid DNA with the enzymes EcoRI and NheI was made to make sure that the enzymes work correctly if given enough time to operate. However it was decided that the contents of the digest mixture would be altered slightly to assure complete digestion:

• 4ul of EcoRI
• 4ul of NheI
• 10ul of pGFP-rrnB DNA
• 4ul of FastDigest buffer
• 28ul of sterile distilled water

Instead of allowing just 1 hour under incubation these digests were given 2 hours to digest under incubation. The resulting digest solution was then placed in the freezer for storage.

Further digests of the pGFP-rrnB DNA

In addition to carrying out this confirmation digest, we digested pGFP-rrnB plasmid DNA with EcoRI and NheI enzymes but on an individual basis. We prepared 10ul each for the 2 different enzymes and had them stored in the -20C freezer.