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aharring 12-17-2018 08:42 PM

Few questions about minion
I want to obtain long read data for 6 bacterial isolates. I have purchased the minion starter kit with the ligation kit and native barcode kit. I am confused on why the protocol requires the Blunt/TA ligase master mix and the NEBNext Quick Ligation Module, it seems like its redundant. Could I just use the NEBNext Quick Ligation Module which does blunt and TA ligation for both barcoding and adding the adapter?

From what I understand is that I do the dA tailing on my DNA, ligate unique barcodes to each sample, pool the different barcodes together at a certain concentration and ligate the adapters.
Last question, how necessary is the qubit? I only have a nanodrop and was curious if anyone has prepped a library using a nanodrop only.

aharring 12-18-2018 12:23 PM

So ONT contacted me with an answer to my question.
The Blunt/TA ligase master mix was validated in-house for the ligation of the barcodes. However, feel free to just use T4 from the Quick Ligation Module. If you go this route, please see the following suggestion:

So, the total reaction would include:

End prep DNA 22.5 Ál

Native Barcode Adapter 2.5 Ál

NEBNext Quick T4 DNA Ligase 10 Ál

NEBNext Quick Ligation Reaction Buffer (5X) 15 ul

Total 50 Ál

I plan on skipping the FFPE repair step because I plan preping fresh gDNA from bacteria cultures. Has anybody tried this before?

gringer 04-07-2019 02:48 PM

Qubit or Quantus (or other similar fluorescence-based methods) are pretty much essential for nanopore sequencing. The nanodrop has issues with RNA contamination, and gives concentrations that can be a couple of orders of magnitude higher than the true DNA concentration.

You can make do with just a nanodrop, but you need to take a lot more care with making sure that the DNA is as pure as possible. I wouldn't recommend nanodrop-only when starting out with nanopore sequencing.

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