[Anne Mette Fiil Eskildsen]

Hi Tim.

I tried a protocol that incorporated all your suggestions (proteinase K digestion, isopropanol in binding buffer, chaotropic salt and/or EtOH in wash buffers) and got a much better yield!

It was still lower when extracting from plasma than when extracting from pure water, but that is probably to be expected considering the protein content and other potentially disruptive components of the plasma.

I took a look on the BOMB protocol #7.1 as well, and have a question regarding the different drying conditions for silica and carboxyl beads. What is the reason that silica beads should be thoroughly dried at 50 degrees while carboxylated beads should only be dried briefly at RT?
I know that drying is important for the complete removal of EtOH, but do not understand the reason behind the different drying conditions

Kind regards, Anne

• This reply was modified 3 months, 3 weeks ago by Anne Mette Fiil Eskildsen.

[Anne Mette Fiil Eskildsen]

Hi Tim

Thank you very much for your answer! I really appreciate your inputs 🙂

Can you explain me why it is not a functional DNA extraction method? The “protocol” (including buffer recipes) was supplied together with the silica particles from the manufacturer. It is intended for purification of viral DNA from plasma, but I thought it might work on a standard DNA ladder as well (although these fragments are much shorter)

 

Regarding the wash buffer: as i understand, it is the low pH of the wash buffer that keeps the DNA associated with the beads during the wash step. But I will try including some chaotropic salt or EtOH in the buffer

 

Regarding the protein content of plasma: that might be an issue yes, it would also explain why the recovery is fine from water but not from plasma. Currently, I do not perform proteinase K digestion, as it is not part of the protocol, but I will try including a digestion step before the bead binding step to see if it can increase recovery

 

I do also not add isopropanol during the bead binding, my experience is that adding isopropanol/EtOH often leads to beads being super hard to work with (clumping and impossible to resuspend), maybe because of protein precipitation? But I could try to add it to the binding buffer and see what happens

 

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