My samples are infected teeth and i got my samples cryogenically powdered. I want to extract DNA from these samples and then use pyrosequencing analysis, however there are some issues. The company which will serve 454 Pyrosequencing procedures requires at least 50 ng/µL concentration. In our last call they said if i get higher than 5-10 ng they will perform lineer amplification. But i could not increase DNA.
In order to increase concentration, we elongated our incubation times from 30 to 60 minutes both at 37°C and 56°C. In every 15 minutes i vortexed samples and put back in bath. But the concentration values did not change. I am describing our extraction protocol below:
We started by preincubating samples in 250 µL lysosyme buffer for 30 min at 37°C. After incubation Fermentas ThermoScientific Kit DNA extraction protocol was followed. 180 µL digestion buffer then, 20 µL Proteionase K were added, homogenized and incubated for 30 min at 56 °C. 20 µL RNAz solution was added and incubated for 10 min. 400 µL 50% ethanol was added and supernatant was transferred to column. Following washing procedures elusion buffer was added in order to izolate DNA.
What else should i do to increase DNA concentration?
In order to increase concentration, we elongated our incubation times from 30 to 60 minutes both at 37°C and 56°C. In every 15 minutes i vortexed samples and put back in bath. But the concentration values did not change. I am describing our extraction protocol below:
We started by preincubating samples in 250 µL lysosyme buffer for 30 min at 37°C. After incubation Fermentas ThermoScientific Kit DNA extraction protocol was followed. 180 µL digestion buffer then, 20 µL Proteionase K were added, homogenized and incubated for 30 min at 56 °C. 20 µL RNAz solution was added and incubated for 10 min. 400 µL 50% ethanol was added and supernatant was transferred to column. Following washing procedures elusion buffer was added in order to izolate DNA.
What else should i do to increase DNA concentration?