conversion of total DNA (ng) into nM

[HGENETIC]

Hi all, I've just finished preparing 72 samples for sequencing, these have prepared using index primers and so I want to pool them together before running on the sequencer. I've been told I need 10ul of a 10nM pool for each run but I'm not sure how to convert my individual concentrations (currently in ng/ul) into nano Moles? Also, I've quantified my final libraries using pico green, do people think this is an accurate way to quantify them?

Thanks for any help!

[krobison]

You need to know the length of your DNA; here is one useful cheat sheet from Epicentre with example calculations & the useful constants (650 Daltons/bp, for example)

[HGENETIC]

Originally Posted by krobison

You need to know the length of your DNA; here is one useful cheat sheet from Epicentre with example calculations & the useful constants (650 Daltons/bp, for example)

Thanks krobison, I've looked at that site and it's still not clear to me and when I try to calculate the numbers I get very strange results. I have 1ug of a 1.5Mb region and I need 10nM for sequencing, according to the epibio sheet:
1 pmole of 1,000 bp DNA = 0.66 µg so if I have 1.5Mb thats 1500X 1,000bp
1 pmole of 1.5Mb of DNA = 990 ug and I need 10nM so thats 10,000X 1 pmole

There is something I'm doing totaly wrong in my calculations but I cant see it, can anyone enlighten me? Thanks in advance...

[NextGenSeq]

There is no way your library is 1.5Mb in length. What size did you shear it to and what is the average size on the Bioanalyzer? Thats the length you use.

[HGENETIC]

Originally Posted by NextGenSeq

There is no way your library is 1.5Mb in length. What size did you shear it to and what is the average size on the Bioanalyzer? Thats the length you use.

That makes sense now, our library is about 350bp so now the calculations all seem to be within realistic quantities. Thank you very much NGS I thought I was going mad?

[SeqR&D]

Originally Posted by HGENETIC

Hi all, I've just finished preparing 72 samples for sequencing, these have prepared using index primers and so I want to pool them together before running on the sequencer. I've been told I need 10ul of a 10nM pool for each run but I'm not sure how to convert my individual concentrations (currently in ng/ul) into nano Moles? Also, I've quantified my final libraries using pico green, do people think this is an accurate way to quantify them?

Thanks for any help!

Okay, I'm not sure that anyone answered your question.

500ng/uL * 1x10^6 uL/L * bp mol/660g * 1/350bp = 2,164.5nM

ssDNA is 330g/bp mol

Pico Green is a great way to quantify, if you have the time. But, what it cannot do...just as nanodrop and other absorbance type quant methods...is quant your material that is "clusterable". That is...it is not telling you the quantity of library that you have that has both adapters/primers attached, so be careful when you use anything other than qPCR. But, you would assume that after PCR you would have outcompeted all the rest.

[HGENETIC]

Originally Posted by SeqR&D

Okay, I'm not sure that anyone answered your question.

500ng/uL * 1x10^6 uL/L * bp mol/660g * 1/350bp = 2,164.5nM

ssDNA is 330g/bp mol

Pico Green is a great way to quantify, if you have the time. But, what it cannot do...just as nanodrop and other absorbance type quant methods...is quant your material that is "clusterable". That is...it is not telling you the quantity of library that you have that has both adapters/primers attached, so be careful when you use anything other than qPCR. But, you would assume that after PCR you would have outcompeted all the rest.

Thanks SeqR&D, it's a bit daunting the first time you do something like this as after all the work in preparing the sample you don't want to screw it up at the last stage. Much appreciated..

[theduke]

See this too, gives prety much the same result as SeqR&D:

http://www.ambion.com/techlib/append...alculator.html

[giampe]

Hi all,
does anyone know why there is no more the tool of conversion in the Ambion site (http://www.ambion.com/techlib/append...culator.html)?
I need a tool for convertion ng to nM to prepare a run of sequencing of small RNA libraries.
can you help me?
thanks

[SeqR&D]

depends on if it is dsDNA (660g) or ssDNA (330g), and assuming you have a volume associated with your DNA. ng/uL * (bp*mol)/660g or 330g * 1/(length of DNA in bp) * 1e^6 uL/1L. this leaves you with nM.