User:Uriel E Barboza Perez/Notebook/Identification and isolation of pathogenic Bacteria from milk samples

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Identification and isolation of pathogenic Bacteria from milk samples

This work was done during the semester( January 2015-May 2015) at the Molecular Diagnostics Lab of our campus as part of our biotech courses

Contents


Team

People who worked on this project:

Project Description/Abstract

The main goal of this project is only for academic purposes only.

Background

Objective

Hypothesis

Justification

Materials

Protocols

Gel Electrophoresis

  • Gel preparation:
  1. For 1% agarose gel (say 200ml), add 2g of agarose powder to 200 ml of 1x TAE buffer (obtained by diluting 10xTAE stock buffer)
    • Note: The shorter the DNA strand lengths, the more concentrated the gel will be.
    • Use 75-100ml of buffer for preparing one gel.
  2. Heat the mixture in the microwave until the powder has completely dissolved stirring the contents every so often.
  3. Transfer the solution into a disposable container.
  4. Gel stains should be added when the agarose becomes cool enough to touch.(For SYBR Safe gel, add 5μl to 50ml TAE buffer)
  • Electrophoresis setting:
  1. Ensure electrophoresis chamber is clean and dry, tape the sides (with Autoclave tape, NOT standard masking tape) to make watertight. Slot in the desired comb.
  2. Pipette a small amount of the tepid gel mixture around the edges of the taped regions to seal the chamber.
  3. Add remaining gel solution to the chamber, and wait to set. The comb can then be removed from the chamber.
  4. Fill the electrophoresis apparatus half-full with 1x TAE buffer solution (for good electrical contact) and place the set gel in the buffer. Ensure that there are no air bubbles (particularly in the wells created by the comb).
  5. Add the ladder solution to the first well, and the DNA samples to subsequent wells. A loading dye may be added to the mixtures to aid visualisation when loading into wells.
  6. Connect the electrodes to the apparatus (the right way round!). Set DC voltage at 80V (with current at approximately 3 mA) and run for 30-60 minutes (or until the DNA has separated sufficiently).
  • Tips for a Successful Gel:
  • Add buffer, not water, when making the gel
  • Seal the gel mould using autoclave tape (not masking tape) and with hot agarose
  • After boiling buffer and agarose, let it cool before pouring into mould to prevent leakage
  • Use running buffer to lubricate removal of mould else risk breaking the wells
  • High salt is bad so dilute sample after enzymatic reactions
  • Use full volume of well
  • Check DNA is running towards the positive/cathode/red pole
  • Check that your voltage and current are appropriate; running gel too fast will distort the bands
  • Use fresh buffer for each gel, as a pH gradient will build up during each run

Methodology

Lab Notebook/log

January

Notes

This work was done during the semester( august 2014-Dic 2014) at the genetic engineering lab of our campus as part of our biotech courses. If you want more info on our protocols or if you wish to have this plasmid to do this experiment for your classes feel free to contact me at uriel_94@hotmail.com



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