Team:Newcastle/Labwork/26 August 2009

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* 4ul FastDigest buffer
* 4ul FastDigest buffer
* 15ul distilled water
* 15ul distilled water
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'''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.
'''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.

Revision as of 11:42, 27 August 2009

Contents

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
  • 1ul of each Fast digest enzyme
  • 10ul DNA (miniprepped)
  • 2ul 10X Fast digest buffer#

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.

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

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.



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