Cord Blood DNA extraction
DNA extraction from the cord blood samples was performed using the
Mammalian Extraction DNA kit (Life Technologies, Carlsbad, CA) according
to the manufacturer’s instructions. Enzimes Proteinase k and RNAse A
were added to 200 μl of sample and briefily vortexed. Protein digestion
was realized by adding Purelink Genomic Lysis Binding Buffer and then
the mix was vortexed and incubated at 55°C for 10 minutes. Ethanol was
added and mix was vortexed for 5 seconds. Supernatant was column
filtered and Wash Buffer 1 was added and centrifuged for 1 min at 10.000
rpm. The latter step was repeated once added by Wash Buffer 2.
Thefiltrate was discharged and centrifugation repeated. Elution Buffer
at a volume of 100 μl was added to the filter and centrifuged for 2
minutes at 15.000 rpm.
The DNA product was maintained at -4 ° C, at the DNA Bank from Molecular
Biology Laboratory . After extraction, electrophoresis in 0.8% agarose
gel stained with Blue Green was performed and DNA fragments were
visualized by UV light through the transilluminator, to ensure the
method was successfully accomplished.