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Qiagen column eluted DNA gives higher concetration than the original sample


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#1 Fluoresce

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Posted 09 February 2013 - 09:08 PM

Hello everyone!
Does anyone know why this is happening? I need help!
I spiked 2 ug of DNA into 1 ml plasma or TE. Used Qiagen circulating DNA kit to re-extract this sample. Meanwhile I extracted samples with TE alone and Plasma alone to control. Eluted all samples in 100 microlitter of elution buffer. Sampels were diluted 4 times in TE then quantified using PicoGreen assay (96 well fromat), where 100 ul of diluted sample was added to one well followed by 100 ul of PicoGreen reagent. 8x dilution factor.

Standard curve was made using the picoGreen Standard. Also a standard curve using the sample DNA above was created (not much of difference observed). Prepared a 2 ug/ml sample of DNA above in TE and quantified using PicoGreen in the same plate (2x dilution).

Quantified unknown extractions as well as un-extracted DNA uisng the standard curve created with PicoGreen assay. Here I had exactly 2000 ng/ml concetration for the unextracted DNA sample in TE. However, the extracted samples either in plasma or TE showed a huge concentration ~3000 ng/ml and ~6000 ng/ml respectively?!!!!
I have already repeated this twice and each time I get this wierd concetrations in my extracted samples...My mind has gone blank now...Can anyone please explain why this is happening?

Thanks!

#2 jerryshelly1

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Posted 09 February 2013 - 09:34 PM

Thats weird. Run a gel with some HindIII ladder to double check concentrations. Maybe your calibration is off.

#3 Fluoresce

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Posted 09 February 2013 - 09:48 PM

Hi...Thanks for your reply! Yes, that's the plan...Will run the gel first thing this coming week. Standard curve was tested by the un-extracted DNA samples and they are fine. There is something going on when I put that 1 ml spiked sample into the column and elute it in 100 ul of the elution buffer and quantify it uisng PicoGreen...if anything, I should expect less concentrated sample as the Qiagen column can not have a 100% yield... :-/

#4 jerryshelly1

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Posted 10 February 2013 - 09:52 AM

Good luck. I haven't had any problems with Qiagen extraction. I usually get 80-90% of sample from original.

#5 bob1

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Posted 10 February 2013 - 02:29 PM

I would postulate that the concentration has gone up because the eluted volume is smaller?

#6 Fluoresce

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Posted 10 February 2013 - 06:00 PM

@JerryShelly1...Thanks! Do you do anything particularly different than what is suggested by QIAgen? (i.e. warm up the elution buffer or longer incubation of elution buffer in the column?

@Bob1...Thanks for your reply...This was one of my first thoughts...assuming I have eluted exactly 100 ul off these columns, if I re-dilute the sample back to 1000 ul, then the concentrations will make more sense...However then I have to deal with columns low yield. But still I can't imagine how I can go 3x bigger than the original concentartion. Is such thing possible at all?

#7 bob1

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Posted 10 February 2013 - 06:55 PM

I would expect a column DNA extraction (especially for pre-extracted and spiked DNA) to be about 30% of total - if you eluted in 1/10th the original volume and you have 30% yield - that will give you 3x the concentration, but only 1/10th the volume so they total yield is less...

#8 jerryshelly1

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Posted 10 February 2013 - 07:21 PM

I follow kit with a few exceptions. I allow equilibration buffer to sit in column longer, I spin down ethanol at maxi speed for 2x time suggested, I allow elution buffer to sit in column for ~5minutes, I then rerun same elution buffer back through column. Qiagen is a good kit, following the protocol should yield good results.

Bob has a good point. Make sure your dilution calculations are correct.

Good luck

#9 Fluoresce

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Posted 10 February 2013 - 08:06 PM

@ JerryShelly1: Good point to look into calculations but they have been checked so many times. I don't think anything is wrong there. Great tips on how to improve the yield. I will apply your hints from now on. Thank You! Posted Image

@ Bob: I agree! I talked to Qiagen tech support and they also told me that Qiagen columns for reasons that are not known to them, does not do well with naked (pre-extracted) DNA, they suggested to spike the DNA into the buffer (ACL) that will carry the carrier RNA [I don't apply the carrier RNA due to the downstream assays incompatibility]. They say this buffer will protect the DNA than just spiking it into plsma or buffer. I also like he way you explained the higher DNA concentration...I think I'm geting somewhere! Posted Image In fact, when I calcualte the yield the way I expalined above, I will end up with only 30% yield in a couple of DNA samples that I tested. Thanks a lot for your help!

#10 CJVH

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Posted 11 February 2013 - 03:21 AM

Hi
Some thoughts about your problem. Think about the amounts of DNA and not the concentration. When you spike you add 2ug (or 2000ng) of DNA. So the concentration is 2000ng/ml or 2000ng/ul. However when you elute, you do so in 100ul so the concentration is now 2000ng/100ul (not 1000ul anymore) if you get 100% recovery. This is equal to 20000ng/1000ul. So at this stage you dilute 4 times so the concentration changes from 2000ng/100ul to 2000ng/400ul (or 5ng/ul ->5000ng/ml, which is still a total of 2000ng of DNA is you have a total of 400ul. So the 3000ng/ml is not a problem since it represents 1200ng of DNA in a total volume of 400ul. (I must admit I'm not sure about the role that the dilution with the picogreen solution plays, if you dilute your standard and sample the same, the answer should be the same, I think). (However, the 6000ng/ml represents about 2400ng of DNA given a total of 400ul of elution volume which is a problem as you so rightly said)

something else that can come into play as well is the true elution volume. If this is a spin column, you could be losing a few microliters in the column, so the volume you collect can be as low as 80ul - so then the dilution factors will change a bit.

One other thing, what volume did you use to add the 2000ng of DNA. If you were adding 1ul of a 2000ng/ml stock it is very possible that the amount of variation in you pipet is causing a huge change in the amount of DNA that you add.
Hope this helps
Carel

#11 Fluoresce

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Posted 12 February 2013 - 12:00 AM

Hi Carel, This is very nice of you to take the time to help. Great way to approcach the calculations. I think you are rihgt. I'm going to have to check a few things to make sure nothing is coming from contaminants low 260/280 ratio for example (However unlikely). I also need to run the gel, I am a lttle afraid of the conformational changes of larger DNA fragments (i.e gDNA samples). The fluorescent molecules access to DNA fragmetns (Specially the way PicoGreen binds to DNA) can also be a factor. I know two out of three samples I tested had issues with degradation. I still have to run the gel and check the bands.

My pipetted volumes were above 5 ul, it's unlileky that this is the issue. I agree on the elution volume collected from columns. It's hardly 100 ul, however it's usually around 90-95 ul. Thanks again for your help. I really appreciate it.




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