DNA concentration increased

[MonaE]

Hi,
My sample concentration after PCR was 52ng/microlitre. After purification with beads it read 63,2 ng/microlitre. Is this normal? usually we lose some DNA after AMpure and this is the first time it happen to me. Can any one tell me what happened? should I repeat my Qubit measurement?

[dpryan]

Are the before and after volumes the same?

[woshiliangliang]

Maybe something's wrong with your measurement.
You should try some other test methods (like Agilent 2100) for a validation.

[MonaE]

Originally Posted by dpryan

Are the before and after volumes the same?

Yes, the before and after volume is the same

[MonaE]

Would the number of PCR cycle affects?

[addyblanch]

Be aware that temperature of the Qubit reagents can alter results. So if you kept the tube in the Qubit housing or even in your hand longer or at all, it could cause a change. Also if you made up the reagents separately on those occasions they might be slightly different ratios.

Another thought - was your PCR product centrifuged and mixed prior to your first reading? The DNA might not have been homogeneous within the sample, or even pipetting technique could be the answer.

[MonaE]

now I have measured the concentration of my samples after AMPure for two different genes and I got variable reading that I do not know how I can pool them.
Gene A Gene B
ng/μl Quantity in ng ng/μl Quantity in ng
1 17.38 434 0.498 12.45
2 8.92 223 0.904 22.6
3 5.36 134 0.588 14.7
4 13.8 345 0.578 14.45
5 21.2 424 1.242 31.05

[MonaE]

Originally Posted by dpryan

Are the before and after volumes the same?

Hi, I have measured the concentration of my samples after AMPure for two different genes and I got variable reading that I do not know how I can pool them.
Gene A Gene B
ng/μl Quantity in ng ng/μl Quantity in ng
1 17.38 434 0.498 12.45
2 8.92 223 0.904 22.6
3 5.36 134 0.588 14.7
4 13.8 345 0.578 14.45
5 21.2 424 1.242 31.05

[addyblanch]

The table isn't that easy to follow, but from what I can tell is you have lost DNA. Quite a lot in fact. 17ng/ul to 0.4ng/ul is significant. It might be a faulty batch of SPRI beads. We had a similar problem and a new batch solved the issue.

[MonaE]

Sorry for the table it is a system problem. In fact the 17ng/ul is the conc of sample in gene A and the 0.4ng/ul is the con of sample in gene B.

I suspect the beads as well because my first experiment went much better. On the other hand the number of cycles in the PCR varied between the two genes. Could this be the reason? the gene with more cycles give higher concentration.

[addyblanch]

I thought it was an issue of concentration before and after a SPRI clean up?

If its a question about 2 separate PCR reactions it might be that you are not amplifying anything in your second reaction then.

What concentration of DNA are you putting into the PCR?

[MonaE]

The DNa product are with a high concentration but I have diluted them to 8ng/µl, this is according to the protocol requirements. I used 25 cycles with PCR A but 20 cycles for PCR B. The 20 cycles worked very well with my previous experiment. I am afraid I might not be able to repeat these samples because I have a very limited primers material. That is why I wonder if there is a solution for this problem.

[addyblanch]

What volume of 8ng/ul DNA have you used in the reaction?

I doubt this is an SPRI bead issue. It sounds like a problem with your PCR. Have you run a positive and negative control? It might be an issue with either your primers for gene B or the DNA you have put into the reaction.

[MonaE]

I put 5 ul DNA in a total of 20 ul PCR reactions. Before PCR the concentration was between 5,4-8,54 ng/ul . After the beads they really varied.

[addyblanch]

So 40ng per reaction. I'd stop worrying about the beads. Its more likely the PCR. To be honest you're not getting great amplification from any reactions.

Take sample 5 in gene B for example if you put in 40ng after SPRI clean up you have 31ng. The PCR isn't working.

I'd check your PCR reagents. If your primers have worked well before just make a fresh batch, if not it might be worth designing new ones.

[MonaE]

I have measured the samples after the PCR and they had good measurement especially with gene B. After the beads some of them their concentration even increased. The PCR is a custom panel primers that are ready designed and have been tested before and worked very well.

[addyblanch]

If you can post the concentrations before and after clean up.

[addyblanch]

Also you said your pre/post clean up volumes were the same you're eluting into 25ul. 5ul more than the PCR reaction. This would account for a ng/ul concentration difference but not total. The minimum suggested volume for SPRI elution is also 30ul.

[MonaE]

For example one of the samples was 52 ng/ul after PCR then for gene A after purification it changed to be 18,64 ng/ul. another sample was 22 ng/ul after AMPURe became 33,4 ng/ul however when I repeated the measurement now it is 8,92 after AmPure. The other problem is that the differences between the concentration in different samples is so variable between samples and with different genes. Example sample 9 is 17.38 ng/ul with gene A and 1.542 ng/ul in gene B. Could I still pool them? or run them in different chips?

[addyblanch]

Your ng/ul will be different pre and post clean up because of the volume difference. You have a 20ul PCR reaction and a 25ul clean up elution.

PCR won't amplify the same with different primers!