Reducing use of NEB reagents in Nanopore barcoding (96) genomic DNA kit

[liaoyunshi]

Dear All,

Recently I used the nanopore 1D PCR barcoding (96) genomic DNA kit to construct the multiplex library. And I found that I need to use the standard volume of NEB reagent for the End prep and Ligation of Barcode Adapter of each of the 96 samples. I wonder if it is really necessary to prepare each of the 96 samples in a way that likes preparing the single library? I think I can somewhat reduce the reaction volume of each sample as they 96 samples would be pooled together finally.

So I want to know if someone have similar experience on this?

Thanks.

[Edinburgher]

I also want to reduce the volume of my reaction size to save costs since only 1/100 of the final products will be loaded to the sequencer. I guess the only way to validate it is to do a side by side comparison study. It is totally worth it if you need to sequence a lot of samples.

[liaoyunshi]

Originally Posted by Edinburgher

I also want to reduce the volume of my reaction size to save costs since only 1/100 of the final products will be loaded to the sequencer. I guess the only way to validate it is to do a side by side comparison study. It is totally worth it if you need to sequence a lot of samples.

Hi Edinburgher,

Quite happy to hear about people thinking the similar things as me, I even hope to change the reagent brand as the NEB is quite expensive.

[Edinburgher]

Molecularly, it should work. However, validation studies need to be in place before you decide to cut the reaction size. RNA access kit is about $14K for 96 reactions. If one can manage to survive with half of the size, imagine how much he/she can save? I am also surprised by the fact that only a tiny bit (0.1-0.2 ul out of 30 ul) is needed for the actual sequencing.

[liaoyunshi]

Originally Posted by Edinburgher

Molecularly, it should work. However, validation studies need to be in place before you decide to cut the reaction size. RNA access kit is about $14K for 96 reactions. If one can manage to survive with half of the size, imagine how much he/she can save? I am also surprised by the fact that only a tiny bit (0.1-0.2 ul out of 30 ul) is needed for the actual sequencing.

I totally agree with you, I cannot imagine why we should prepare 96 samples respectively in a way for a single sample library, and finally you need only 1/96 of each sample to be pooled.