As we are performing method validation for our DNA extraction protocol we are facing issues.

Sampling Procedure :

Inside of the cheek, swab with a little bit of strenght 10-15 secondes while turning the swabs.

+ avoid eating or drinking 30min before the sampling procedure.

Extraction Procedure :

1) Cut the swabs and put in a 1.5 mL eppendorf tube.

2) Add 200 µl PBS

3) Add 25µl ProtK

4) Add 200 µl BQ1 (lysis Buffer)

5) Vortex for 20s

6) Incubate 70°C 10-15 min

7) Add 200 µl d’EtOH 100%

8) Vortex

9) Pipet the ~ 600 µl into the column.

10) Then continue with standard protocol, centrifuge for 1min at 11'000rpm

11) Discard, wash with Buffer 350µl BQ2 (wash buffer).

12) Centrifuge 3min at 11'000rpm

13) Discard then add 50 µl of TBE (Elution Buffer) heated at 70°C

14) Centrifuge 1min at 11'000rpm

DNA quantity expected : between 20-50 ng/uL

We only have between 1 and 7 ng/uL

How can we improve the quantity of DNA extracted ?

The DNA quantity measurement is performed on Denovix QFX Fluorometer using the DSDna High Sensitivity Assay.