[James Andrew Bagley]

Hi All,

Thanks for making this forum, I love the idea and the project. We’ve been using magnetic beads for our minipreps for a while now but for our use case contamination with even very small amounts of DNA can cause issues downstream. We’ve been trying to find a way to either break down the genomic DNA or do a size selection afterwards as the trace genomic DNA seems to be present regardless of preparation method/lab personnel, but reading through the 4.1, 5.1 and 5.2 protocols it occurred to me that the protocols are similar enough that perhaps a size selection step could be inserted into the plasmid extraction protocol.

This is what I’m imagining it would look like, starting after the addition of N3 buffer

1. centrifuge to pellet cell debris
2. transfer 500 uL supernatant to new plate
3. Add Ethanol to bind genomic DNA to beads (250-350 uL? would need to be determined experimentally)
4. add magnetic beads
5. mix
6. bind beads & transfer supernatant containing plasmid DNA to new plate (save for later)
7. Add EB to beads to elute genomic DNA from beads
8. Immobilize beads and discard EB
9. Refresh with New EB & discard again
10. Transfer supernatant from step 6 back onto beads
11. Add additional ethanol (150-250 uL)
12. continue with plasmid extraction as normal

Does anybody have any thoughts on the feasibility of this before I give it a shot?

[admin]

Ahoy,

In a way I hope you already solved the challenge and I am therefore too late.

But maybe not so I figure I may as well share my thoughts.
I do not know the exact composition of Qiagen N3 but since it is a silica binding buffer I would assume several things that may not be ideal for your purpose.

• DNA (Including Plasmids) is already precipitated -> spinning may lead to some loss.
• The precipitated DNA in N3 will be charged and therefore sticky -> changing tube leads to loss.
• Adding ethanol at this point might lead to salting out and general precipitation of unwanted contaminants.

I do not understand why you would presume that you would be selectively binding plasmids in the procedure you describe.
All in all I think what you propose wont work but maybe you have already tried it and can prove me wrong?

However, could you consider simply using Exonuclease 5 (V)?
From personal experience ExoV is amazingly good at chewing up anything non circular.

Cheers

Tim M

[Author]

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