22 Feb 2012

Bacterial DNA Taking out for PCR

Some polymerase chain impulse (PCR) applications for instance gene prognosis or perhaps typing, need little purified DNA and may even possibly be performed together with crude bacterial extract. Several approaches are actually described just for this course of action along with a number of them are straightforward and also are comprised purely boiling bacterial cells in water. When it reaches this probability, we would like to reveal the extraction way of bacterial DNA for PCR that works well having Gram-positive bacteria such as staphylococci, enterococci, and also Clostridium difficile.


·         Tris-EDTA (TE) buffer. 10mM Teis –HCL, 1mM ethylene tetraacetic acid (EDTA), pH 8.
·         Lysis buffer: 50 mM Tris-HCl, 25 mM EDTA, 250 U involving lysozyme per milliliter, pH 7. 5. Include 15 U regarding lysostaphine per milliliter intended for staphylococci.
·         Digestion solution: 0. 6% Nonidet P40 (Sigma Chemical, St. Louis, MO), 0. 6% Tween 20, 0. 6 micrograms of proteinase K for every milliliter.

Methods of Bacterial DNA extraction for PCR are:

  1. Increase bacteria on appropriate agar or even liquid medium.
  2. Harvest tissue through the culture medium and resuspend these individuals inside TE buffer to have an optical density connected with 2. 0 at 580 nm.
  3. Transfer 1mL on this suspension into a microtube as well as wash the tissues double using TE buffer by centrifugation (2000g).
  4. Resuspend the pellet within 500 microliters associated with lysis buffer in addition to incubate at 370C pertaining to 1 h.
  5. Add 500 microliters regarding digestion solution and incubate at 560C regarding 1 h.
  6. Inactivate proteinase K by warming the actual microtube at 950C pertaining to 10 min.
  7. Store the DNA preparation frozen at -200C in aliquots.

The actual regarding DNA preparation added to the PCR mix must not go over 1/20 on the remaining size mainly because at a decrease ratio, the EDTA seen in the particular getting ready may inhibit DNA polymerase activity. The proper ratio depends within the bacterial species as well as PCR applications and therefore should be optimized.

See more: www.bioteachnology.com

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