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-   -   Reducing use of NEB reagents in Nanopore barcoding (96) genomic DNA kit (http://seqanswers.com/forums/showthread.php?t=80162)

liaoyunshi 01-17-2018 05:01 PM

Reducing use of NEB reagents in Nanopore barcoding (96) genomic DNA kit
 
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 01-17-2018 11:27 PM

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 01-17-2018 11:36 PM

Quote:

Originally Posted by Edinburgher (Post 214167)
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 01-17-2018 11:44 PM

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 01-18-2018 12:13 AM

Quote:

Originally Posted by Edinburgher (Post 214169)
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.


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