Good day, currently I am practicing extracting total DNA using drosophila melanogaster (fruit fly) adult as well as larvae as my sample with Qiagen DNeasy Blood and Tissue Kit. However, I keep obtaining DNA with smears.

My main purpose is to extract total DNA and send the extracted DNA for 16S Amplicon sequencing (V3-V4 region). The insect is from the Lepidoptera family and I will only be extracting DNA from the larvae (which is like caterpillar).

I followed this protocol:

1) Grind 50 mg tissues by disposable micropipette tips a 1.5 ml micro-centrifuge tube with added 180μl PBS

2) Add 200 μl buffer ATL + 20 μl of proteinase K and vortex and incubate at 56˚C for 1 hour

3) Add 200 μl Buffer AL + 200 μl absolute ethanol, vortex

4) Centrifuge 8,000rpm 1 min

5) Pipet the supernatant in DNeasy Mini spin column

6) Centrifuge for 1 min at a speed of 8000 rpm, Discard the collection tube and flow-through, place the DNeasy Mini spin column in a new tube

7) Add 500 μl Buffer AW1 and repeat centrifugation.

8) Add 500 μl Buffer AW2

9) Centrifuge at a speed of 12,000 rpm for 4 minutes

10)Place DNeasy Mini spin column in a 1.5 ml tube and add 50 μl buffer AE and incubate for one min at room temperature followed by centrifuge at a speed of 8000 rpm to elute the DNA

So my questions are:

1) Is there any suggestion why this happen or is there any method to decrease this degradation

2) How "intact" should the DNA be/how little degradation should there be to be sent for NGS?