HUNT Extraction and concentration normalisation of DNA

HUNT Extraction and concentration normalisation of DNA

HUNT biobank is experienced in procedures involving extraction of DNA from human blood (clotted blood,  whole blood, packed blood and buffy coat) and saliva.

Primarily by the Gentra Puregene Blood Kit (Qiagen), DNA has been extracted from all samples collected for HUNT2.

For blood samples (buffy coat) collected for HUNT3 and HUNT4, DNA-extractions have mainly been done using the MasterPure DNA purification Kit for blood and Buffy Coat DNA Purification Protocol (Epicentre). Both extraction methods are based on desalting and are both validated by the supplier and HUNT biobank.

DNA yield

DNA yield varies dependent on quality and number of cells in the sample. For whole blood, theoretical yields are expected in the range of 8-50 µg DNA per ml whole blood. By experience, average DNA yields at the HUNT Biobank are ~ 20 µg DNA/ml blood (±10). Fresh samples are expected to give higher yields and our experience is mostly based on long-term stored samples (- 80 °C).

DNA yield are controlled routinely by measuring every eight sample spectrophotometrically.

Normalisation of DNA concentration

At normalisation, DNA is diluted to give concentrations of e.g. 50 or 25 ng/µl dependent on the original sample concentration. At HUNT Biobank, Biomek pipetting robots are used for normalisation. Before normalising to a given concentration, all concentration measurements are performed fluorometric using PicoGreen reagents.

After normalisation of samples to 50 ng/µl, a mean concentration of 50.19 (± 9.9) ng/µl has been shown (based on the measurement of 1255 samples).

DNA – quality / purity

Quality / purity (ratio A260/A280) are controlled routinely by measuring every eight sample spectrophotometrically.

For samples of good quality A260/A280 is expected to lie in the range of 1.6 – 2.1. Lower values may indicate protein contamination while higher values may indicate a high content of RNA. At HUNT Biobank the average ratio A260/A280 in general is found to be 1.8 (±0.2).

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Contact

 

 

Address
HUNT Research Centre

Forskningsveien 2
7600 Levanger

Phone
Our phone is not manned during the summer holidays (16/7-16/8).
+47 74 07 51 80 | +47 74 01 92 40 +47 916 83 253 

Email
kontakt@hunt.ntnu.no