Hi there,
I've got these bacterial samples and were to extract DNA for posterior 16S DNA sequencing. I have two groups of samples: group A are isolates with nothing than bacteria cells (I hope =) )and group B are the same samples, but silicate was added to the isolates. I extracted DNA with the DNEasy power soil kit (Qiagen) and the yield for group A was about ~40 ng/uL meanwhile for group B it was too low, about 0.5 ng/uL. So, my guess is that silicate is somehow messing my extraction yielding almost no DNA.
(I do have a hint indeed, that DNA is being captured by flowing silicate before the final step of the extraction, which is passing through a silica-column. So DNA would be trapped before the collumn.)
Could anyone provide me with a protocol, product or a hint for removing silica from samples prior to DNA extraction?
Thanks in advance,
Leo
Centrifugation should do the job, nevertheless, try to use phenol:chloroform method.
Hi Abhijeet,
Wouldn't centrifugation drop down the cells alongside the silicate to the pellet?
What do you think is the size of your silicate particles? I guess, they must be heavier than the cells. So it can form pellet before the cells in a light centifugation step. If the particles are too small, then in the phenol:choloroform method (after cell lysis), the DNA would be in aqueous phase and it can be collected as supernatent to process further.
That makes sense. I don't know their size but I do know they are >90% of the mass of these samples. I am going to try light centrifuging it and quantify. If it still doesn't work I'll go for the phenol:chloroform protocol. Thanks
I am incomplete agreement that a low speed spin should take out the silicate particles, they should clump in the early extraction stages. It might take a little trial and error to get the correct rpms for the centrifuge to take out the silicates and leave the cells but this should do the trick. Good Luck. It would be interesting to se what is in this first pellet of presumed silicates. I think the easiest way to confirm this is to see if they are slectively soulibilized in an HF solution. Only HF should dissolve the silicates., HCl should leave them intact.
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