DNA isolation from bloodspots

Acc.no: 1V9FG4S | Published: 2010-03-20 by jennie

Keywords: leukocytes, DNA, bloodspots, blood, erythrocytes, sickle-cell, newboorn

Blood contains cells with a nucleus (leukocytes) and cells without a nucleus (erythrocytes). DNA is isolated from the leukocytes by boiling of bloodspots

Required Materials

Methanol
Mini centrifuge , Eppendorf-centrifuge 5840 R
Blood , EDTA-treated
Gloves , disposable
Eppendorf tubes
Tweezers
Scissors
Pipette , disposable
Blotting paper , 3MM
Milli-Q water
Water bath , Thermocycler 60

Instructions

Method

One drop EDTA-blood is placed on blotting paper using a disposable pipette (approx. 50μl) and left to dry.

Cut an approximately 2*2mm spot (use scissors, tweezers and disposable gloves) and transfer the spot to an eppendorf tube.

Fixation: Add 2 drops of methanol to the spot in the eppendorf-tube and leave for 1 - 2 min. Pour out the methanol and evaporate for 5 min in a vacuum centrifuge.
Methanol binds the proteins to the blottingpaper.
Boiling: Add 100μl MilliQ water, boil for 15 min at 95°C (leave old spots to boil for 15 min). Centrifuge for 15 min at 13000 rpm (4°C).
Troubleshooting

Old bloodspots: Boil for longer time. New bloodspots: Make sure they are not too big Methanol should not be decanted.