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[Turnerac0987]

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!

[phage434]

I would definitely verify that there was DNA to start with.  Do this with a gel, not with the spec.  The spec can give false readings showing DNA when there is none, and you could be purifying those contaminants out of the solution, leaving nothing.  Save your flow through and washes from the protocol, and run those on a gel to check if you are washing the DNA out of a column accidentally.