PCR PURIFICATION DETAILS
An amount of 200 µL of binding buffer and 200 µL of the pooled PCR product
were inserted into a purification column from the GeneJet kit and mixed by
pipetting (for less than 200 µL of PCR product equal amount of binding buffer
was used). The centrifugations were performed at 13.200 x g as follows: first
centrifugation for 1 min and then the column was removed from collection tube,
the buffer was discarded ad the rim of the collection tube was dried; 700 µL of
wash buffer from the purification kit was added to the column and then the second
centrifugation for 1 min was performed following the same operation as the first
one; centrifugation for 2 min was then employed to remove all residual ethanol;
30 µL of elution buffer from the purification kit were added, then the column
remained at room temperature for 1-3 minutes and then centrifuged at 13.200 x g
for 2 min; finally the purified DNA was stored at -20 °C. The purified DNA was
then fragmented using the following reagents kit: UNG enzyme 1U/µL – UracilDNA-Glycosylase 1U/µL – Fermentas, Fast-AP™ enzyme 1U/µL Thermosensitive Alkaline Phosphatase 1U/µL – Fermentas, Fast-AP buffer 10 x Fermentas. In order to process one sample, the work solution was prepared as
follows: 1.2 µL UNG enzyme, 1 µL Fast-AP enzyme and 4 µL 10x Fast-AP
buffer.