Influence of EDTA and magnesium on DNA extraction from blood samples and specificity of polymerase chain reaction

  • H Khosravinia
  • KP Ramesha
Keywords: EDTA, DNA extraction, Mg2 concentration, PCR.

Abstract

This study consisting of two trails conducted to examine the impact of initial EDTA level added to blood samples on quantity and quality of genomic DNA isolated from avian fresh blood and the influence of
initial EDTA level with various levels of MgCl2 added to polymerase chain reaction (PCR) final volume on amplification pattern. EDTA level added to collected blood samples had no significant impact on
quantity as well as quality of extracted genomic DNA. However, higher levels of EDTA increased the OD260 and enhanced the OD260/OD280 ratio beyond 1.8-1.9 which is broadly accepted as an indicator of high quality DNA. To avoid such an error, EDTA level in initial blood sample must not exceed 9 ìg/ìl blood. The initial amount of EDTA has no influence on PCR process if a valid DNA isolation protocol is used. Addition of MgCl2 from 1.0 to 2.4 ìl in a final volume of 25 ìl could support the amplification properly. Low levels of MgCl2 results in incomplete amplification but levels higher than 2.4 ìl impedes the amplification by negative interference to the whole reactions.
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