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Falsely High Yields on Qiagen Miniprep? Assayed by Nanodrop


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#1 harukosama

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Posted 16 May 2012 - 04:52 PM

Hello,

I am trying to solve a mystery that has plagued me for a long time. Your help is appreciated.

The fundamental problem is a low yield after a restriction digest on plasmid, purified by PCR Purification. I will start out with 4ug plasmid, purify, and end up with 500ng, meaning on the order of a 15% yield.

Another fact is that digesting and purifying a PCR product is higher yield, at 70-80%.

The plasmid is isolated via Qiaprep spin column. It is a high copy plasmid in DH5a E coli in 4mL of LB. I grow it from a colony or frozen stock for 10-16hours. I get about 10ug of plasmid this way. (Changing the culture time doesn't change much).

The PCR product is isolated via either gel extraction or PCR purification (this does not affect the % yield from digest).

All DNA concentrations are measured by Nanodrop.

I've done all sorts of troubleshooting. I've tried washing multiple times with PE; heating up EB to 60deg and incubating the column at 60deg for 5-10minutes; tried adding QG to wash out agarose;spun the column after PE wash for extra long to get rid of all ethanol.

This low yield is consistent for me across all sorts of different plasmids, but I've only used DH5a.

This low yield happens when I run the digest on a gel and gel extract (10-20% yield). I don't remember trying this on a digested PCR product. Other people in my lab report similar yields.

On a gel running the plasmid by itself, I do not see much of a genomic contamination and only slight hints of RNA low-MW smear http://i.imgur.com/1cvJW.jpg. In this image I also tried to quantify the amounts in the bands. The ladder is from NEB (http://www.neb.com/n...roductn3200.asp). From left to right, the 4 bands are supposed to be at 1ug, 750ng, 500ng, and 250ng. What I can tell is that the right most band is probably less than 125ng, but more than 50, which means my miniprep yield appears to be much higher than what I really have.

So my questions are:
-Is there ever a case where I can get falsely high yields on Miniprep spin columns? How do i prevent this?
-In this case, would this lead to a deceptively low yield on restriction digest? Why would this happen?

Looking forward to your feedback-

#2 phage434

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Posted 16 May 2012 - 05:39 PM

Some possibilities: The RNAse in your P1 buffer was not added, or is dead. Lots of RNA can occur with little visible on a gel, since it is so diffuse. Gu-Cl from P2 can be carried over, which absorbs like DNA. Make sure you wash not just the bottom of the column but also the ledge. In general, I trust gels, not nanodrops. You may be overloading the column, which can result in genomic DNA coming through. Also, vortexing after P2 and before spin down after N3 will shear genomic DNA. Since, again, the bands are diffuse, you may not see how much there really is on a gel.

#3 harukosama

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Posted 16 May 2012 - 09:35 PM

Thanks phage - what's your opinion on an order of magnitude estimate of my yields here? http://i.imgur.com/1cvJW.jpg

The ladder is from NEB http://www.neb.com/n...roductn3200.asp

I don't really have much practice quantifying this way.

#4 phage434

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Posted 17 May 2012 - 06:48 AM

Why is the ladder resolution on your gel so poor? Usually gels show a nice ladder and poor bands, you have the opposite. Is this a 0.5% gel? The gel does not appear to show much RNA or gDNA contamination, but I would try to make the resolution better. Perhaps it just needs to be run longer. If you are plannng on quantitation on the gel, I would use a loading dye that eliminates xylene-cyanole blue and bromcresol blue, leaving only the orange G, to get rid of the masking effect of the dye on the strength of the bands.

Have you measured a DNA standard (such as an NEB pUC19 sample at 1 ug/ul, diluted) with your nanodrop and compared to a gel? Your nanodrop might simply be calibrated poorly, or you could be having bubbles in the drop. Do you use 1 ul or 2 ul loading the nanodrop? I found 2 ul to be much more reliable. Also make sure you clean the pedestals (top and bottom) before use.




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