What DNA extraction method is recommended?

There is no strict request on the choice of the extraction method. The only requirement is the quality of the genomic DNA which must meet with the general recommendation for DNA quality for long-range PCR (A260/280 in range 1.8 – 2.0 and A260/230 in range 2.0 – 2.2). It is users responsibility to validate the assay with their gDNA samples.

We may not recommend using Prepito DNA Blood 400 Kit (CMG-2003) or Chemagic DNA Blood Kit (CMG-1086), NucleoMag Blood 200 µL or MagDEA DX extraction kit as we observed a negative interference with the PCR.

On the other hand, we may recommend the following extraction methods that have been internally successfully tested:

Chemagic DNA Blood KIT (CMG-1086-EFS)from Revvity
QIAamp Blood Mini Kit (250) (51106) from Qiagen
Maxwell RSC Whole Blood DNA Kit (AS1520) from Promega
NucleoSpin Blood (740951.50) from Machery-Nagel

I need to transfer my library to another flow cell due to an unexpected flow cell error. Is there a way how to do it?

In such case, it is possible to recover the library from the flow cell and transfer it to a new one. Please follow this link – Library recovery from flow cell – to read the detailed article about how to proceed.

I realized that I don’t have enough data after the sequencing and the sequencing run is already over. Is there any advice how to rescue my data?

You can always try to run the flow cell again without reloading the library because there still may be some unsequenced library on the chip. Simply keep the flow cell with the library in MinION and set up another run. This will generate a new output folder, however, barcodes will be identical as in the previous run. So you can then simply combine the fastq files of each sample after the run with the ones from the previous run and repeat the analysis.

Refueling of the system with fresh buffer might be requires. As a general rule, we recommend that if the translocation speed drops below 300b/s then this is a good time to consider refuelling. Follow the instruction on ONT Community channel in the article titled “Refuelling your flow cell”.

How stable is the ONT Flush buffer?

The FLUSH BUFFER (FB) vial contains 15ml of FB. The FB contains ATP which is sensitive to freeze/thaw cycles. We recommend to make aliquotes to use only 1 vial each time.

1.Take 12 pieces of 1.5ml tubes,
2.Aliquot 1.17ml from the Flush Buffer (FB) in each of the tubes,
3.Freeze the tubes,
4.Use 1 tube of 1.17ml FB each time you do flow cell priming.

What are the storage conditions of ExoSAP-IT and what is the best practice to repeatedly use the reagent without deterioration of its performance?

We recommended storing ExoSAP-IT reagent at -20 °C in a non-frost-free freezer. It can be stable for up to 2 years.

Unfortunately, we do not have data showing how the stability changes with every freeze/thaw cycle, however, our general advice is to make aliquotes when working with enzymes and minimize the number of freeze-thaw cycles. This way you can mitigate the impact of freeze/thaw cycles to minimum.