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Old 10-25-2011, 09:49 AM   #1
Turnerac0987
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Default NO yield after purification using Qiagen kits - Please help!

Hey everyone,

I've been trying to start a library prep for Next generation sequencing, but I haven't been able to get past the first purification step using a QIAquick MinElute Purification kit.

My starting material is amplified PCR products containing around 100 amplicons per sample, and the concentrations are around 50ng/ul. As far as I can tell, it is very high quality PCR product.

The first step in my library prep is a MinElute purification, and after I tested the quality on the nanodrop I realized that I lost ALL my DNA! There is absolutely NO curve and the concentration is <5ng/ul with highly variable 260/280 ratios (between 1.3 and 2.4) I also tried running the products on a Gel and saw no bands.

Once I figured out I was losing all my DNA somewhere in the purification, I started troubleshooting. I used a completely different kit, the QIAquick PCR purification kit, and got the same results. This should rule out the columns being the problem. Also, the ethanol for the PE buffer was added by a different person in the PCR kit, and I also double checked that I used the right kind of ethanol for the MinElute kit, so I really don't think the ethanol or PE buffer is the problem.

I made sure everything was the right pH during this whole process by using pH indicator and it was all fine. I also tried centrifuging for a longer period and higher speed. Finally, I ran the purification on a QIAcube to rule our human error and again got NO DNA.

I've done around 12 purifications now trying to figure out the problem but I'm getting the same results. I'm really at a loss about what to try next to figure out this problem. My boss is reluctant to try a different purification method yet because the protocol we're following says to use the
Qiagen MinElute kit.

If anyone has ANY ideas about why I'm losing all my DNA during purification it would be greatly appreciated! Even if it's something I've already tried, I'll try it again.

Thanks so much everyone!
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Old 10-25-2011, 11:47 PM   #2
Cofactor Genomics
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Hello,
What are the size of your amplicons? Did you check them on a gel prior to running on a minelute? Did you have a positive and negative control reaction and did they behave as expected? The answers to these questions would help to troubleshoot.

Another method, which is completely compatible and acceptable is to use a mag-bead cleanup, such as Ampure. Without knowing the size of your amplicons, it is a little hard to troubleshoot or offer suggestions. The other option would be to use a Microcon cleanup. I have seen MinElutes fail (as well as Microcons), however in my case it was usually user error.

If you offer up more information, we may be able to help.

Jon
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Old 10-26-2011, 05:10 AM   #3
pmiguel
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Yes, on what are you basing your contention that these are "very high quality PCR products". You only mention using a Nanodrop -- that would be confounded by many factors. Did you run the PCR products or pools on a gel or bioanalyzer chip prior to subjecting them to purification?

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Old 10-27-2011, 06:12 AM   #4
Turnerac0987
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Sorry for this late response. Here is some more information on my samples. My lab sent 16 DNA samples to Raindance to be amplified with a targeted library. The samples they sent back were analyzed on a bioanalyzer and were shown to have amplicon yields between 450 and 750 ng, and the length of the amplicons are between 150 and 600bp.

They only sent us 10ul of product back, so we're reluctant to use any of it to run gels, especially since they already sent us back a report of the sample quality.

Once I realized that I lost all of my DNA somewhere during purification using these samples, I started troubleshooting using some other random PCR products. I test these samples on nanodrop before and after doing purification, but I'm going to switch to testing them on a gel. I realize nanodrop is not the best way to test DNA quality, but I just figured that if I started out with a curve before purification and ended up with nothing afterwards that something was going wrong.

Let me know if this is enough information. I'm still sure I'm losing my products during purification, but to further make sure I'll start running everything on gels.

Thanks for your help.
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Old 10-31-2011, 07:16 AM   #5
Cofactor Genomics
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If you really believe it is the column that is causing the problem, then simply purify a 100 bp ladder, in parallel with your sample (or just use the ladder separately). This will serve as a positive control. Purify, wash, elute, run out on a gel to see if it is there.
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Old 10-31-2011, 08:02 AM   #6
Turnerac0987
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Update: After doing some more tests, I'm pretty sure that I'm not in fact losing *all* my DNA, just most of it. I tested some PCR product that started at 400ng/ul. After doing purification, the concentration was 25ng/ul. Is it normal to lose 90% of your sample when doing purification?

Even though the protocol I'm using says to use Qiagen MinElute columns, I'm going to look into using ampure beads instead. I can't imagine that method being any worse than the results I've been getting with the columns.
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Old 10-31-2011, 08:38 AM   #7
pmiguel
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Depending on how that concentration was measured, you may just be losing other stuff that absorbs at 260 nm. (Primers, nucleotides, etc.)

Ampure is a good method, though.

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Old 11-01-2011, 12:11 PM   #8
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Turner,
You also might want to contact Raindance for technical support. They should be able to recommend possible approved changes for their protocol.

I am used to seeing somewhere between 70 - 90% recovery from a minelute cleanup.
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Old 02-06-2017, 07:30 AM   #9
KimUTA
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We had failures due to wrong pH for column binding. Did you include the pH indicator solution? You might need to adjust pH with sodium acetate.
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