Landscape of Interacting Microbes - DNA Extraction Protocols
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Pelleting bacterial cultures
- Grow a pure colony in 5mL of liquid broth
- Upon visible growth and pellet at the bottom of the tube, centrifuge the culture at 1,700 RCF for 5 minutes (preferably using a swing-bucket rotor)
- Remove the supernatant, then vortex and homogenize the pellet and transfer to a 1.5 mL/2mL microcentrifuge tube
- Centrifuge the microcentrifuge tubes at the highest speed for 5 minutes
- Remove the supernatant and use the pellet for DNA extraction
- Pellet can be stored at -80C
- Add ~250 uL of beads to a screw-top microcentrifuge tube with O-ring seal screw cap
- Vortex to homogenize the pellet, then add the mixture to the microcentrifuge tube
- Add 600 uL of RLT buffer (without beta-mercaptoethanol) from Qiagen’s AllPrep DNA/RNA Mini kit to the microcentrifuge tube
- Bead beat at maximum speed for 10 minutes
- Extract DNA following the protocol for Qiagen’s AllPrep DNA/RNA Mini kit
- Elute DNA in 30-100 uL elution buffer and quantify using Qubit
RNAse treatment and DNA purification
- Bring DNA sample to 100 uL
- Dissolve RNase A in DNase/RNase-free water or TE to a stock concentration of 10 mg/ml
- Add enough 10 mg/ml RNase A to the sample for a final concentration of 10-100 µg/mL and mix well
- Incubate at room temperature for 15 minutes
- Purify DNA using Zymo Research’s Genomic DNA Clean & Concentrator-10 kit
- Elute DNA in 30-50 uL elution buffer (target concentration is >50 ng/uL)
- Quantify and assess the quality of purified DNA using Qubit, NanoDrop, and by running >50 ng of the DNA on a 1% agarose gel
- Send 20 uL of >50 ng/uL DNA to Eurofins Genomics for bacterial genomic sequencing on the Nanopore platform