SEQanswers

Go Back   SEQanswers > Applications Forums > Sample Prep / Library Generation



Similar Threads
Thread Thread Starter Forum Replies Last Post
Sequencing of Epstein-Barr virus genome in a mixture of total human DNA hinkwok Genomic Resequencing 0 06-26-2011 11:47 PM
How much total RNA for a transcriptome? Dangermouse 454 Pyrosequencing 13 05-10-2011 05:32 PM
tophat total alignment zorph Bioinformatics 4 12-09-2010 04:09 AM
PubMed: Chloroplast genome sequences from total DNA for plant identification. Newsbot! Literature Watch 0 08-28-2010 10:30 AM
Successfull DNA conversion by EZ DNA Methylation-Gold kit??? nedoluzhko Sample Prep / Library Generation 1 07-21-2010 09:30 PM

Reply
 
Thread Tools
Old 09-15-2010, 02:05 AM   #1
HGENETIC
still a novice
 
Location: Cardiff

Join Date: Jul 2010
Posts: 34
Default conversion of total DNA (ng) into nM

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!
HGENETIC is offline   Reply With Quote
Old 09-15-2010, 04:51 AM   #2
krobison
Senior Member
 
Location: Boston area

Join Date: Nov 2007
Posts: 747
Default

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)
krobison is offline   Reply With Quote
Old 09-15-2010, 05:32 AM   #3
HGENETIC
still a novice
 
Location: Cardiff

Join Date: Jul 2010
Posts: 34
Default

Quote:
Originally Posted by krobison View Post
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 g 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...
HGENETIC is offline   Reply With Quote
Old 09-15-2010, 05:55 AM   #4
NextGenSeq
Senior Member
 
Location: USA

Join Date: Apr 2009
Posts: 425
Default

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.
NextGenSeq is offline   Reply With Quote
Old 09-15-2010, 07:45 AM   #5
HGENETIC
still a novice
 
Location: Cardiff

Join Date: Jul 2010
Posts: 34
Default

Quote:
Originally Posted by NextGenSeq View Post
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?
HGENETIC is offline   Reply With Quote
Old 09-15-2010, 04:01 PM   #6
SeqR&D
Member
 
Location: San Diego

Join Date: Sep 2010
Posts: 25
Default

Quote:
Originally Posted by HGENETIC View Post
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.
SeqR&D is offline   Reply With Quote
Old 09-16-2010, 01:01 AM   #7
HGENETIC
still a novice
 
Location: Cardiff

Join Date: Jul 2010
Posts: 34
Default

Quote:
Originally Posted by SeqR&D View Post
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..
HGENETIC is offline   Reply With Quote
Old 09-17-2010, 04:16 PM   #8
theduke
Member
 
Location: San Antonio

Join Date: Aug 2010
Posts: 10
Default

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

http://www.ambion.com/techlib/append...alculator.html
theduke is offline   Reply With Quote
Old 09-05-2012, 11:10 PM   #9
giampe
Member
 
Location: Bari, Italy

Join Date: Aug 2009
Posts: 21
Default

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
giampe is offline   Reply With Quote
Old 09-06-2012, 02:59 PM   #10
SeqR&D
Member
 
Location: San Diego

Join Date: Sep 2010
Posts: 25
Default

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.
SeqR&D is offline   Reply With Quote
Reply

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 03:18 AM.


Powered by vBulletin® Version 3.8.6
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.