( eliminate the heme-containing red blood...)Genomic DNA Extraction from Buffy Coat Using the Perfect gDNA Blood ,,, Mini Kit

eliminate the heme-containing red blood cells. The buffy coat layer is difficult to see and pipette. It is viscous and is present as a thin milky layer above the lower red blood cell layer. Although separating the blood into its components requires an additional step, the benefits of higher gDNA concentration and yield out-weigh the extra effort.

Materials

• Perfect gDNA Blood Mini Kit
  

Additional items needed:

• Ethanol (95100%); pipette tips; Eppendorf microcentrifuge; Eppendorf Thermomixer set at 900 rpm and 70C
• Whole blood samples from three individuals were collected in Vacutainer (Becton Dickinson tubes) containing three different types of preservatives: EDTA, Sodium Heparin and Sodium Citrate

Method

Several experiments were carried out to validate the performance of the kit when using buffy coat. Volumes ranging from 20 l to 200 l of buffy coat were taken from an entire vial (3 ml) of blood and tested for handling, yield, concentration, and purity as well as downstream applications such as PCR1. The method outlined below provides a step-by-step procedure for separation of buffy coat and isolation of gDNA from either a whole vial of blood or an aliquot of blood.

Entire vial of blood:
The term vial refers to the tubes typically used for collection of the blood. Vacutainer (Becton Dickinson, USA) is an example of this type of vial. These vials are usually glass and may hold various amounts of blood with 1.5 ml being the minimum amount required for this procedure.

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