- This topic has 4 replies, 4 voices, and was last updated August 14, 2020 at 11:31 am by .
- Posts
Dear BOMB team,
I found this website and I would like to use your protocol (#7 DNA extraction), but I want to ask you a few questions before to use it:
1) Is this protocol works for other bacteria (most of them gram-positive bacteria)?
2) I have the MagneSilParamagnetic Particles (https://www.promega.com/<wbr />products/nucleic-acid-<wbr />extraction/genomic-dna/<wbr />magnesil-paramagnetic-<wbr />particles/?catNum=MD1441) which are are silica-paramagnetic particles. Do you think that I can use these beads for the protocol (#7 DNA extraction)?. Only the protocol says “Carboxyl-coated or silica-coated magnetic beads” but does not provide more information.3) Do I need to do an extra step to “prepare” these beads (MagnesilParamagnetic Particles) before using them?
4) Instead of Sarkosyl, Could I use SDS or Triton at the same concentration?
5) I have been having problems with the yield and also with the contaminants that cause to get at the end low yield and dirty DNA. I saw that in this protocol described the quality control (260/280) but I can not see the 260/230 relation. Is the 260/230 ratio above 1.8?
Thanks in advance for your help.
Best,
Hi Karina,
sorry for the late answer! Everything’s a bit crazy right now ^^
1) we never checked this, but I would assume it works with gram+ bacteria too. If not, one could think about digesting the cell wall before the lysis. We did something similar with yeast (#6.5).
2) Both bead-coatings should work with that protocol, although I personally prefer the silica-coated ones. I never tried these commercial beads, but don’t see why they wouldn’t work. However, other people had a few issues using commercial carboxyl-beads with our buffer systems. I would just give it a try or do a quick synthesis of BOMB-beads: https://bio-protocol.org/e3394
3) That depoends on the beads and what the supplier information says to do before using them. Our beads are simply suspended in water at a ratio of 1:1 wet mass volume to water volume.
4) I would again say, give it a try. We never tested this as far as I remember, but I don’t see why SDS shouldn’t work. Then again, I’m not a Chemist by training ^^
5) If you check in the original publication (https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3000107) at Fig 2I our 260/280 ratios for gDNA are between 1.75 and 1.90, so yes, a bit higher is sometimes the case.
Hope it helps and sorry again for the delay. If you have any more questions, feel free to ask. I checked the notify button in this thread now.
Hi Phil,
No problem for the late answer! I perfectly understand this hard situation…
Thanks so much for all the information! I will test your protocol when this pandemic passes.
Stay safe,
Best,
Karina
Hi everyone!
Can I perform blood sample extractions with this protocol?
If someone tested it, they would like to know the sample volume used and relevant observations. I intend to use this methodology to extract DNA from the swine blood virus.Thank you in advance for your help.
Best regards,
Ahoy, Not to long ago I was wondering the same thing and I did a quick trial with my own blood. In my single attempt I did not succeed in extracting enough to be visible on a gel without PCR. Talking to my colleague afterwards he suggested to use far less input than I did. So from my point of view I’d have to say, not without modification or figuring out input concentration first.
Cheers
Tim M
- You must be logged in to reply to this topic.