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Old 10-19-2009, 03:16 PM   #1
tamosan
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Location: USA

Join Date: Apr 2009
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Default illumina primer/adaptor concentrations

Hi all,

I want to switch from illumina supplied library prep kit to assembling my own. I've been seeing helpful threads about primer/adapter sequences, but cannot find any information about their concentrations.

I am specifically interested in concentration of SE lib prep kits:
PCR primers 1.1/2.1
Adapter mix

Does anyone know these concentrations provided in the kit (or point me to threads containing these information)?

Thanks!

Last edited by tamosan; 10-19-2009 at 04:17 PM.
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Old 10-29-2009, 06:12 AM   #2
Exeplex
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Location: Exeter, UK

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Default Oligo concentrations

Quote:
Originally Posted by tamosan View Post
I am specifically interested in concentration of SE lib prep kits:
PCR primers 1.1/2.1
Adapter mix

Does anyone know these concentrations provided in the kit (or point me to threads containing these information)?

Thanks!
The concentration in the adapter mix is 15 uM - you can find this info in the Cronn et al 2008 paper in NAR, although you can also work it out from the Illumina protocol: for DNA fragmented to an average size of ~500bp, 5 ug in 10ul gives a total of 15 picomoles; therefore for a 10:1 molar ratio, you must be putting in 150 picomoles of adapter (also in 10ul).

For the PCR primer concentration, the thread "Excess PCR primers in final product " has posts indicating the stock PCR primers to be 25uM; these are used 1 in 50 in the enrichment PCR, i.e. at a final concentration of 500nM.
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