library concentration

[Tarie]

Hi,
I wanted to ask if, as regards the concentration of the library, are there any scores to understand if it's okay? I use the ' nextera flex for enrichment ' protocol with MiniSeq sequencer .
for the quantification , I use the Qubit dsDNA BR Assay Kit.
thank you for the attention .

[Ben3]

Hello Tarie.

Just for clarification, are you asking for the normal concentrations for a library? If so, that could be variable but I would say you should have at least several ng of material by the finish with something like nextera flex.

What concentrations are you getting from the Qubit? Also, are you checking them through other methods like qPCR, Bioanalyzer, or Tapestation?

[Tarie]

I don’t use other methods to check the concentration of the library .
my perplexity arises from the fact that in the last runs I obtain high library concentration values of about 17 ng/ul, then I proceed with the diluition phase at 1 nM as required by the protocol and denaturation but the cluster density that I have in the end is always low about 140 K/mm.
Before I obtained libraries of about 8-10 ng/ul and I always had excellent cluster density .
I can’t understand what it could be because I use the same protocol , the same thermal cycler and th same condition.
furthermore the concentration of the samples before the pool are good .

thanks a lot for the attention and the help

[Ben3]

Tarie Thanks for the explanation. I understand what you mean.

So, I've had similar issues in the past. I'm not sure how to narrow it down, but I can tell you a few methods that I did.

1. Whenever my final library concentrations were high, I noticed it was much more inaccurate during the loading and dilution. So I diluted them in half, checked them on the Qubit, and used the new concentration before starting the loading process. In your case, your concentration is 17 ng/ul. Next time I would take the libraries and dilute them in half to 8.5 ng/ul before proceeding with the pooling and loading steps. But check them on the Qubit first to make sure it's actually 8.5 ng/ul and not 6 or 7 ng/ul. It could be that the Qubit isn't super accurate, or the pipettes aren't as accurate, or a number of things. But it worked better when I diluted it to a lower concentration to start sometimes.

2. I would also look at your last few runs and see if any of the other cluster densities dropped or only the ones where the library concentration was high. I would notice my cluster densities dropping and it was either that I needed new NaOH or maybe some of my reagents were old.

3. My third recommendation is to see if there's another way to check the concentration. I've usually had good luck with Qubits but a few times my readings weren't accurate and didn't match up with other machines.

Good luck and let me know if you have more problems or questions!

[Tarie]

Ben3 Thanks so much for the advice and the help.

if I may I would like to ask you if to dilute the library you use only RSB or also water DNase/RNase-Free?

thanks a lot

[Ben3]

Tarie no problem. But I just used RSB for my dilutions. My libraries were already in RSB so I kept it consistent.

[Tarie]

Ben3Hello, thank you for your reply.
I wanted to ask you if you also dilute the library to 1 nm and calculation you use to do it ? If after bringing it to 1 nm, you check that it is actually at 1 nm ?
It is normal that if my samples are very concentrated then after the second amplification also the library must be very concentrated?
thanks a lot

[Ben3]

Tarie I only diluted them in half and then started my loading protocol as normal. Also, I didn't check when I brought them down to 1nm. But depending on the protocol, it should be normal that they're really concentrated after the 2nd amplification step.