To extract DNA from a bean beetle for MiSeq sequencing, follow the protocol below. The protocol is based on one used for previous MiSeq studies of the bean beetle microbiome by Dr. Hassan Salem in the lab of Dr. Nicole Gerardo at Emory University.
- Place beetle in 1.5 mL microfuge tube and freeze with liquid nitrogen or in -80C freezer. Add 0.5 ml sterile, distilled water and homogenize with a sterile pestle.
- Spin in microcentrifuge for 1 minute at maximum speed.
- Remove supernatant.
- Add 200 ul lysis buffer and pipet to mix thoroughly.
- Add 100 ul micro-glass beads.
- Add 200 ul phenol:cholorform:isoamyl alcohol (25:24:1, stored at 4C). Make sure tubes are completely closed. You may want to wrap in parafilm.
- Vortex at 4C for 5 min.
- Add 200 ul TE buffer (pH 7.5 or 8).
- Centrifuge for 5 min at maximum speed.
- Transfer 400 ul aqueous (top) layer to a new 1.5 ml tube. Make sure that the pipet tip doesn’t touch the while, gunky interface of the layers. Dispose phenol tube in phenol:chloroform waste in fume hood.
- Extract once more with 400 ul phenol:chloroform:isoamyl alcohol.
- Add 1 ml of 100% ethanol. Mix by inversion.
- Precipitate at -20C freezer for at least 1 hr.
- Centrifuge for 10 min. at maximum speed at 4C.
- Remove supernatant. Allow pellet to dry completely (usually overnight). Residual ethanol will interfere with DNA suspension.
- Before sending for sequencing, resuspend the pellet in molecular grade, sterile filter water and check the DNA concentration with a nanodrop. The DNA concentration of all of your samples should be standardized to a given concentration. We standardize to a concentration of 50 ng/ul.
1ml Triton X-100
5ml 10% SDS
1ml 5M NaCl
500ul 1 M Tris pH 8
100ul 0.5M EDTA
For sequencing, we have used Mr. DNA Lab (http://www.mrdnalab.com/) for 16s Illumina MiSeq sequencing, because they are willing to sequence a small number of samples at a time. In addition, they will carry out preliminary analysis of the data through their data analysis pipeline free of charge.