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Does anyone have trick or tips on maximizing DNA yield from tiny samples?

I am currently extracting DNA from tiny insect samples and am looking for a way to maximize my yield. (My extractions are working, but am looking to optimize as my current yield may limit the number of msat loci I am able to use).

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  • James Smith · Thorne Diagnostics Inc.
    Can you add some carrier DNA that won't interfere with your analysis?
  • Tom Jones · University of Essex
    What extraction protocol are you using?
  • Paula Dias · Université de Rennes 1
    maybe increasing concentration with 10M buffer during the extraction?
  • Juliane Casquet · Paul Sabatier University - Toulouse III
    I had the same problem when working with very small spiders (1-2mm): I wanted to use only a few legs to be still able to do some taxonomy and the yield I obtained was too low. Then I developped a very easy DNA extraction protocol (attached) with no filtration to minimize the quantity of DNA lost during this step. Using this kind of protocol could help you to increase your DNA yield.

    The other possibility would be to use a centrifugal evaporator such as a SpeedVac. By evaporating part of the water (or buffer) in your samples, it quickly increases their DNA concentration. Good luck!
  • Jess Vickruck · Brock University
    @ Tom: Good question! At the moment I am using the Qiagen DNeasy kit with the exception that after lysing I do the binding step twice and then elute 50uL twice for a total volume of 100uL.
  • Jess Vickruck · Brock University
    @ Juliane: This looks great. I'll definitely give it a try! Thanks :)
  • Doug Bintzler · DNA Analysis, LLC
    One trick we used when increasing DNA yields would be to warm the elution buffer or water to around 37C. To increase concentration we would follow this with ethanol precipitation. Using a speedvac to reduce volume and increase concentration is okay if the samples were eluted in water. I would give caution to evaporating samples eluted with buffer since this also concentrates the salts in the buffer.
  • Silke Werth · University of Iceland
    Perhaps you could try a kit for DNA extraction from forensic samples. These kits tend to have tiny membranes and are better for working with smaller sized volumes.
  • Tom Jones · University of Essex
    I have found, when using kits like this, that leaving the elution buffer to sit in the tube for 1 - 2 minutes before spinning it down often increases the DNA yield.
  • Mohamed Elasmar · ain shams university ,faculty of medicine
    Speedvac to concentrate your sample as suggested by Doug could be a simple answer to your problem
  • Jean-Nicolas Audet · McGill University
    Maybe you are already doing this, but I found that leaving 100% ethanol (or isoprop) for precipitation for ~1 hour @ -20°C helps a lot for high DNA yield. Also minimizing wash (eihter not doing it or doing it very quickly with ice cold alcohol) helps. As another person suggested, resuspending in warm buffer (37°C) also helped in my case.
  • Doug Bintzler · DNA Analysis, LLC
    Good idea Tom. We have actually let the elution buffer sit up to 5 minutes before spinning to collect the sample.
  • Christopher Hamm · University of California, Davis
    I had 2mm butterfly wing clips to work with during a project and had good success by tweaking the Qiagen DNEasy kit protocol. 1) be sure to really macerate the samples; 2) extended incubation with proteinase K (I typically went ~12 hours); 3) use half the recommended amount of elution buffer AE, 4) warm elution buffer AE to 60C and let that solution sit on the membrane for 10 minutes. I hope this helps.
  • Nathan White · Anglia Ruskin University
    Assuming you are using a Qiagen based extraction, when you carry out the final elution simply use less buffer, although you will have less DNA sample it will be of a much higher concentration. This really depends on what downstream applications you depend on doing with your sample(s).
    Hope this helps.
    -Nath
  • Kateřina Rylková · Czech University of Life Sciences Prague
    I was isolating DNA from slime swabs with Qiagen DNEasy Blood and Tissue Kit, following tissue protocol. I increased the yield by long lysis period - usually overnight (16 hours or even longer) + multiple vortexing and by prolonging of binding period of AE buffer for several minutes.
    Thank to all for another tips.
  • Valentine Kheng · Columbia University
    You can also do whole genome amplification. We use GenomiPhi V2 DNA Amplification kit to increase our DNA samples using 1 uL of the extracted DNA.
  • Patrick Mardulyn · Université Libre de Bruxelles
    We have tested several methods of DNA extraction for tiny insects (Culicoides imicola) and even smaller nematodes (Caenorhabditis remanei), and found that a quick, simple and cheap protocol gave us the best results: each individual is placed in a 0.2-ml micro tube containing 10 µl of a lysis buffer (Tris-HCl 10 mM pH 8.2, KCl 50 mM, MgCl2 2.5 mM, Tween-20 0.45%, gelatine 0.01%, proteinase K 60 µg/ml) and squashed using a 10-µl pipette tip. Each tube is then incubated at -80°C for 30 min, at 65°C for 1h and at 95°C for 15 min. This extract is directly used for microsat PCR amplifications. For really small organisms, this method worked better for us than Qiagen DNEasy columns, standard phenol/chloroform, or Chelex extractions. And we were able to keep these samples for more than one year in the freezer.
  • Dr (Mrs.) Kamalakshi · Government of Tamil Nadu
    Use kit provided by Qiagen.
  • Helena Kuivaniemi · Geisinger Health System
    If you are using a method with alcohol precipitation, you can add glycogen as a carrier and improve the yields. Glycogen will not interfere with doanstream applications. Whole genome amplification is a good idea before doing genotyping to make sure you have enough material for all genotyping reactions.
  • Kasey Fowler-Finn · University of Wisconsin - Milwaukee
    I am not sure which extraction method you are using, but for amplifying microsats from spider eggs, I used chelex extractions, which yields plenty of DNA. I believe the protocol was 50uL chelex solution, 0.25uL pro-K, incubate (the incubation can vary greatly, but you can find various protocols online) at 55 for 1 hour, then boil for 10 min (to deactivate the proK). Use the supernatant for PCR (the beads interfere with the taq). This method is super fast, really cheap, and, in fact, works best with small amounts of tissue. you will have to clip the tips before pipetting (or the beads don't fit through the pipette). Good luck and I hope you find a method that works well for you!

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