Difference between revisions of "Team:Glasgow/Measurement"

Overview

^[1]

Introduction

• Aim 1
  • Sub-aim 1
  • Sub-aim 2

• Aim 2
  • Sub-aim 1
  • Sub-aim 2

Materials and Methods

Transformations

These were carried out as per our standard calcium chloride protocol.

Calibration

Both the OD600 reference point and the fluorescein fluorescence standard curve were carried out as per the InterLab 2017 Plate Reader Protocol

Dimerisation

To generate dimer plasmids a strain of E. coli JC8679 ^[2] known to cause interplasmid recombination at a higher frequency and so cause multimers was used. Plasmid DNA of each InterLab device was transformed into JC8679 using standard transformation protocol and was then repurified from JC8679 by normal QIAGEN DNA miniprep protocol. The plasmid extracts were then separated by standard gel electrophoresis and the DNA band corresponding to the dimeric plasmid DNA was cut out from the gel and extracted by standard gel extraction protocol a diagram of the gel showing the multimerisation versus the standard monomer plasmid can be seen in figure 2. Multimerisation didn’t occur 100% of the time so this needed to be repeated until dimers were obtained for all of the test devices and the two controls. This extract was then transformed into DH5α E. coli.

Measurements

For the purpose of the measurements two biological duplicates (colonies) of all 6 test devices transformed in to DH5α, along with a positive and negative control, were grown up in LB broth overnight at 37C with shaking at 220rpm. These were then diluted down to an OD600 of 0.02 and 100 l of the samples were placed into wells of a clear bottomed black sided 96 well plate as shown as per figure 3. This was placed into the plate reader at 37C with shaking at 200rpm and measurements of OD600 and Fluorescence at an excitation of 485nm and emission of 530nm were taken at time points of 0hrs, 2hrs, 4hrs and 6hrs.

Results

Discussion

NEB5α

Dimers

References

1. ↑ Kiliç, A. O., Pavlova, S. I., Ma, W. G. & Tao, L. 1996. Analysis of Lactobacillus phages and bacteriocins in American dairy products and characterization of a phage isolated from yogurt. Appl Environ Microbiol, 62, 2111-6.
2. ↑ Summers, D.K. & Sherratt, D.J. Resolution of ColE1 dimers requires a DNA sequence implicated in the three-dimensional organization of the cer site. EMBO J 7, 851-8 (1988).