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I am attempting to elute a 9kb plasmid from a square of filter paper sent to me to be transformed into DH5a E. coli (photo below). Each filter paper square has been spotted with 1ug of plasmid DNA.

plasmid

I have two questions:

  1. AddGene's protocol states to elute the DNA using 30ul of TE (I'm using H20 as that's all I have atm). Using a similarly sized piece of filter paper to test, I added it + 30ul of h20 to a 1.5ml tube with a quick 4k spin down and have observed that the 30ul is completely absorbed by the paper as shown in the photo. How should I resolve this? Add an additional 30ul? I also tried to use a pipette tip to shove the paper down and squeeze out the water but you can see it didn't work.

  2. What volume should I use for transformation knowing that the filter paper contains 1ug and I need to use additional elution volume? Most protocols I've read call for 1-5ul of DNA and AddGene calls for 2ul assuming 30ul elution volume.

Thank you.

elution

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  • $\begingroup$ Are you using the same kind of filter paper (thickness matters)? Did you follow their directions, which say to use 1/2 the piece of filter paper (because they send you a two spots) and to use 30 µL (not the 3 µL you wrote)? $\endgroup$
    – tyersome
    May 22 at 0:55
  • $\begingroup$ My sample isn't from AddGene. And I can only estimate it's the same thickness. It seems to be the same based on close inspection. The spot on the paper isn't marked and so I wouldn't know exactly the boundaries to cut out of the paper. The 3ul is an error - I'll fix that now. I used 30ul. $\endgroup$
    – doremi
    May 22 at 2:38
  • $\begingroup$ 1 ug is a LOT of plasmid, so your elution doesn't have to be very efficient. I would figure out how much water your test piece of filter paper can absorb by adding more and spinning down until you start to see free liquid. I would then use double that volume to elute your actual sample, take 30ul and proceed with your transformation. If your centrifuge is a benchtop eppendorf-style, I'd use the max speed rather than just 4k. $\endgroup$
    – Armand
    May 22 at 7:06
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The answer to this is... some.

You only need a tiny amount of plasmid DNA to transform bacteria like DH5$\alpha$, typically in the 1-10 ng range, but much less, as low as about 10 pg (10 picograms) may still work. If you can get even 0.2 ul of the liquid out of that tube, try using that in the transformation.

Things you can try;

  1. remove the paper, spin the tube down and see if you can recover even a tiny amount of liquid.
  2. Add a bit more liquid - another 30 ul will be fine
  3. Snap the tube lid closed on the paper to hold it in place, then spin the liquid out of the paper - this is quite difficult to do.

Note - even with 10% DNA recovery (very low for this method), with 30 ul you will still have 100 ng of DNA, or about 3.3 ng/ul.

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  • $\begingroup$ These are all excellent suggestions, especially using the lid to spin out the paper. Thank you! $\endgroup$
    – doremi
    May 22 at 22:13
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    $\begingroup$ FYI, I was able to use a total of 60ul of TBE to elute the plasmid on filter paper, using 1ul for my transformation with success. $\endgroup$
    – doremi
    Jun 3 at 20:21
  • $\begingroup$ Good, so you probably transformed a few ng. In my experience smaller amounts <5 ng work better than higher amounts anyway. $\endgroup$
    – bob1
    Jun 3 at 20:39
  • $\begingroup$ Indeed. I did 16 experiments with varying volumes ranging from 1ul to 10ul and the lowest volumes performed best. It was a ~10kb plasmid. I actually posted most of my diy transformation protocol here. $\endgroup$
    – doremi
    Jun 4 at 16:22

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