Hi all, hope you will be able to help me with this. How to calculate fmoles from a given concentration and size of the dsDNA?

I have been struggling to obtain a good read N50 by sequencing genomic DNA on MinION from Oxford Nanopore Technologies with the ligation sequencing kit, and I wonder if it wouldn't be easier (quicker protocol), cheaper (no 3rd party ligation enzymes) and maybe get a higher read N50 using the Rapid kit (less washing steps and DNA loss). Can anyone share your experience? Thanks in advance.

Dear researcher,

After amplifying my target gene (1900bp) into the required cDNA by PCR,>cut specific gel bands and purify the gel and the concentration was 30ng/ul. Next, performed A-Tailing according to the Kit protocol (Tiangen#RT121221); add 15ul (30ul/ul gel-purified product) + 4ul Tailing A reaction Buffer + 1ul Taq DNA polymerase = 20ul.

For the next pMD™19-T vector cloning kit (TAKARA# code number 6013), the solution needs to be prepared by inserting 0.1 pmol~0.3 pmol of DNA,

1- How to convert ng/ul concentration to pmol/ul?

2- How to calculate the amount of insert DNA from the above?

According to manual the formula is

ng = ( x fmol )( N )(660 fg/fmol)(1 ng/10^6fg)

where N is the size of the DNA in base

pairs, and x is the number of fmoles. so , if my entry clone is 2817bp and insert size is 1374bp ,then N=4191bp and x=10. that means i will need 28ng/microliter DNA. Am I right??

Another question, According to manual I have to use 20fmol destination vector and the volume is 1 microliter. Can I use more than 1 microliter DNA say- 2 microliter containing 20fmol DNA? Because my Destination vector DNA is low concentrated.

i believe its 7018.2 g/mole

( 5'- rUrCrA rCrCrG rGrGrU rGrUrA rArArU rCrArG rCrUrU rG -3' )

Please can someone explain clearly how i can convert this?

Hi, our lab is planning to get a MinION sequencer for whole genome sequencing of fungi. We plan to use the long contigs from the MinION in combination with Illumina to construct genomes. I have some practical questions about the flow cells:

1) How long can you store unopened flow cells in the fridge? I am trying to figure out if we can buy a stockpile of flow cells to store and use whenever we need them. On the ONT website it is said they should be used within 12 weeks. Does anyone have experience with storing flow cells?

2) How long can you store a flow cell in the fridge after washing? Should they be used as soon as possible after washing or can you store them for a while?

3) Are there differences in performance between R9 and R10 flow cells?

Especially with regard to whole genome sequencing.

Hi, everyone

I was reading many PCR protocols where I found that 100 pmol = 100uM. I am unable to understand this conversion as I found in google that 1pmol = 1000000 micromol.

can anyone guide me in a detailed way?

Thank you

Considering the use of this kit ( SQK-RBK004 ) for cost efficient sequencing of multiple bacterial genomes per flow cell. Any comments on its efficacy etc. would be welcome! Thanks!

Are nanopore flow cells reusable?

What is the maximum number of run per one flow cell?

I'm working with genotyping of S aureus