Ahmad Mahmoudi - Department of Animal Science, Young Researchers Club, Khorasgan (Isfahan) Branch, Islamic Azad University, Isfahan, Iran
Reza Motafeghi - Department of Animal Science, Young Researchers Club, Khorasgan (Isfahan) Branch, Islamic Azad University, Isfahan, Iran
Maryam Shahriar - Department of Entomology, Young Researchers Club, Khorasgan (Isfahan) Branch, Islamic Azad University, Isfahan, Iran

Objectives: We have compared three different methods extraction of DNA from blood. Many different methods and technologies are available for the isolation of genomic DNA. The purpose of this study was to test three different method for DNA extraction of blood based on existing Protocols for blood DNA extraction. In general, all methods involve disruption and lysis of the starting material followed by the removal of proteins and other contaminants and finally recovery of the DNA.Methods:Materials are need: Extraction buffer Proteinase K, Phenol-chloroform isoamyl alcohol, Chloroform isoamyl alcohol, Ice cold 95-100% ethanol(keep aliquot in the freezer), 70% Ethanol Ultrapure (DNA-& DNase-free) and water TE buffer.Triton X-100, Tween-20 and Tween-80 were bought from sina Gene. Other chemical used were analytical grad (siqma). Whole blood was collected in a vacutainer tube containing 100Ml of 15% EDTA. The simplified on mililieter of blood was treated with an eqval volume of low- salf buffer containing 10 mM Tris-HCl PH 7.6, 10 mM KCl, 2 mM EDTA (TKE) containing 4 mM MgCl2 (TKM). Twenty five microliters of NP-40 was added and the cells were lysed by inverting several times. The suspension was centrifuged at 1000g for 10min at room temperature (RT). The pellet of mostly leukocytes was saved and washed two more times with TKM buffer. The final pellet was resuspended in 0.2ml of TKM buffer. Fifteen microliters of 10% sodium dodecyl sulfate (SDS) was added, and the whole suspension mixed thoroughly and incubated for 5min at 55° C. After adding 75 μl of saturated NaCl ( ??? 6M) , the tube was mixed well and centrifuged at 12000g for 5min. The supernatant containd DNA, wich was precipitted using ethanol. DNA was redissolved in 0.5ml of 10 mM Tris-HCl, 1 mM EDTA, PH 8.0(TE). The yields of DNA were calculated from the absorbance at 260nm (Maniatis et al.,1982) and by comparison with a molecular weight DNA standard on an agarose gel stained with ethidium bromide.Results: Extraction of DNA with either NP-40 or Triton X-100 gave a high yield of undegraded DNA in less than an hour. The concentration of magnesium ion in the buffers was critical to obtaining intact, high molecular weight (HMW) DNA. Greater than 10 mM MgCl2 led to degradation. Addition of EDTA to the buffer inhibits this degradation. Preparation of DNA from blood stored at room temperature or incubated at 37°C for 24 hr resulted in the same amount and quality of DNA as from samples frozen at −70°C. DNA from blood samples that had undergone more than four freeze-thaw cycles was found to be partially degraded. The modified RM can be applied to extract DNA from as little as 10 μl of blood (340 ng of DNA) and from dried blood samples. DNA samples remained intact and undegraded for longer times when DNA was dissolved in higher concentrations of EDTA. Effect of Different Detergents in the Extraction of DNA from Blood Samples. Two kinds of detergent were used to study their effect in the extraction of DNA. One group lysed the red cells, resulting in a clear solution; the other.

DNA extraction, Phenol-chloroform , blood