No single method will suit all microorganisms. One option is to create a single, complex method to cover all bases, but that results in unnecessarily complexity, cost and wastage when working with easier species.

 

We have provided below, a number of methods to suit a variety of bacterial morphologies and substrate types. This is a growing list and we would like to hear from you if you have adapted one of our methods to suit your sample types. Remember too, we are always available to help provide you with a custom method.

There are two optional steps in the procedures:

 

1. A prewash with the WASH solution.

2. The lysozyme and incubation step at 37°C.

 

The WASH solution is an osmotically buffered surfactant that removes polysaccharides and so is intended only for mucosal samples and capsuled bacteria. The lysozyme is only needed for species with tough cell walls.

 

The figure below outlines where the stages should be used.


Method table


Technical Tips

  • The procedure should be optimised for different cell types and different substrates.
  • The procedure can be automated using most liquid handling robots.
  • prepGEM Bacteria is a preparative method for DNA extraction - it is not a purification protocol.  Its purpose is to lyse cells and to strip the DNA of nucleoproteins. The formulation creates DNA that can be used for SNiPs, STRs, quantitative, multiplex and routine PCR applications.
  • If the DNA appears cloudy, it is OK to sediment the solids. However, be aware that high speed spins will sediment the DNA (which is very high molecular weight). Sediment at 2000 g for 2 minutes and aspirate away the DNA without disturbing the pellet.
  • For accurate yield assessment, a qPCR is recommended. The DNA produced by the kit is approximately partially single-stranded. If standard fluorescent chelating dyes are to be used for quantification, this factor should be taken into consideration. See Quantification of ZyGEM extracted DNA using fluorescent dyes.
  • As with any preparative method for nucleic acid extraction, for best results prepare and manage samples at 4ºC, or on ice, before and after extraction.
  • If extracting DNA from samples containing EDTA, CaCl2 should be added to a final concentration of 200 µM to improve the enzyme activity
  • The enzymes is stable for at room temperature but we recommend storing the tube at -20ºC once it has been opened. This is to protect it from accidental contamination.
  • For long-term storage of the extracted DNA, add TE buffer to 1x and store at -20°C.

 

 

Methods using prepGEM Tissue

 

• Extraction from Yeast (Coming Soon)

 

Other Documentation

 

• ZyGEM method optimisation (PDF 799k)
prepGEM Bacteria Application Note (PDF 557k)
prepGEM Bacteria Quickstart guide (PDF 557k)