[Anne Mette Fiil Eskildsen]

Hi BOMB community

I am trying to purify DNA from plasma samples, without the use of a commercial kit, as i want to have insight into the buffers I use. I use commercial silica beads (MagPrep silica particles from Merck) together with the following buffers:

Binding buffer: 5 M GTC, 1% Triton X-100, 10 mM Tris-HCl, pH 7.5

Wash buffer: 10 mM Tris-HCl, pH 6.7

Elution buffer: 10 mM Tris-HCl, pH 8.6

I mix 1x plasma sample with 5x binding buffer, giving a final concentration of ~ 4 M GTC during the binding step.

I spike plasma samples with a DNA ladder before the purification, to allow for easier analysis of the purified DNA. I include a positive control containing nuclease-free water spiked with DNA ladder.

My issue is that the DNA recovery from plasma samples are only 5-6% of the input DNA, whereas it is > 60% from the positive controls. I have performed the purification several times, getting consistent results.

From the literature, it seems that DNA purification from plasma using silica-coated magnetic beads and chaotropic salt is an established method that yields high DNA recovery. I do not understand why my recovery from plasma samples is so low, while it is much better when extracting from water.

Can anyone suggest some ideas for troubleshooting?

Thank you!

 

• This topic was modified 3 months, 1 week ago by Anne Mette Fiil Eskildsen.

[admin]

Ahoy,

I dare say that what you posted in itself is not a functional DNA extraction method.

Unless you are also adding some sort of solvent or additional salt to your wash buffer, there is no reason why your wash buffer wouldn’t wash away the DNA you are trying to capture.

If, the issue isnt in your wash step then i would assume the issue might be with plasma being very high in proteins and quite low in DNA. It could be that you are facing issues with proteins overwhelming the binding capacity of your beads. Silica beads are great at binding DNA but also not very specific, they will bind proteins as long as the right charge difference is given. I take it you are adding isopropanol to allow for binding? If so, how much? Going as low as 35% isopropanol (final c. including binding buffer/beads in solution/sample) could possibly help by reducing protein precipitation.

Lastly, are you proteinase K treating your sample? If not then I would strongly recommend this as a troubleshooting step.

Cheers,

Tim M

[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

 

[admin]

I hope you have had some success? I am unaware of any wash step that does not rely on DNA remaining precipitated (= alcohol present) to stay bound. I would really recommend you just try something like the straight forward BOMB protocol #7.1 :

https://bomb.bio/protocols/   –> Nucleic Acids –> #7.1 DNA extraction.

Cheers

[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 2 months, 3 weeks ago by Anne Mette Fiil Eskildsen.

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