Qiagen column eluted DNA gives higher concetration than the original sample - (Feb/09/2013 )

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!

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

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... :-/

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

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

@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?

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...

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

@ 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!

@ 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 . 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! 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!