Hi, really struggling to prepare a protocol for feather bacterial DNA extraction protocol and wanted to know if I need to add salts at some point to the following (realised that I don't think I'm precipitating the DNA properly):
1. I have 3 feathers as starting material, stored in PBS.
2. Sonicate samples for 3 x 5 minutes (wasn't sure if longer would be better), then vortex thoroughly before removing the feathers (want to dislodge attached bacteria, and also collect free-living which should be in the PBS).
3. Aliquot liquid into 2 tubes; one for gram-negative and one for gram-positive.
4. centrifuge both for 10min @ 13000rpm to pellet the bacteria then pour out the liquid under a sterile hood (couldn't see any pellet but have been told that the bacteria should stick to the tube and so pouring the supernatant out should be fine).
5.for gram-negative bacteria; re-suspend in 100ul buffer ATL (Qiagen kit), add 20ul proteinase K and incubate at 56C (NOT SURE FOR HOW LONG - I TRIED OVER NIGHT BUT WAS HOPING MAYBE 3 would be sufficient). Vortex, add 300ul buffer AL with ethanol and mix thoroughly.
6.for gram-positive bacteria; re-suspend in 150ul enzymatic lysis buffer (which I have prepared as 20mM Tris-HCl, 2mM EDTA, 1.2% Triton-X 100), add lysozyme to 10mg/ul then incubate for 1 hour at 37C. Add 25ul Proteinase K and incubate as above. Add 100ul ethanol and vortex thoroughly.
7. Add the 2 tubes together and mix before pipetting onto a Qiagen spin column and centrifuging at 8000rpm for 5 minutes.
7.wash with 100% ethanol (200ul) and centrifuge again, as above.
allow the membrane to dry completely before eluting in 60ul Buffer AE. leave to elute for ~1hour then centrifuge 2 min on each side at 8000rpm.
- carry out ethanol precipitation/ purification to concentrate the DNA.
Does this sound like it should work to anyone? I don't think it includes a salt precipitation step?
Please help!
Please , Look in attach file, you found DNA extract kit from promage company uses both G- and G
You should just collect your feathers and need to collect bacteria for DNA extraction so just use freshly collected feathers and not preserve it or use PBS in which you have kept as preservative.
After lysozyme and salt addition, we usually perform phenol-chloroform purification step instead of columns. We are getting 100% pure DNA and you can also add Rnase in your final suspension buffer to remove rna contamination. If you want complete procedure, I can share.
Try the new PureLink Microbiome DNA purification kit - it has protocols for soil, stool, etc. works really well for microbiome applications. so just wash off bacteria from feathers and move forward with DNA isolation using this column based kit
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