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Old 02-10-2017, 12:37 PM   #1
JVGen
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Default Nextera XT v2 Index - Oligo Concentrations?

I use Nextera XT v2 Indexes for library prep, but I can't stand the tubes that Illumina provides them in. I'm thinking of ordering from a third party in a plate format, and dispensing into strip tubes. It'll probably be cheaper, too.

Anyways, Illumina won't share the primer concentration in the kit, so I measured in by Qubit ssDNA, and got some varied results. Was curious to know if anyone else has measured them before, or what concentrations others use? I did see someone recommend 5uM stock, which makes sense based on my measurements. However, I'm curious if Illumina has taken the time to figure out optimal index concentration for each unique index. My data suggests either this, or that they're terrible at diluting their primers accurately

I could understand if all i5's were once concentration, and all i7's were another, but I can't explain S505....

Index | (ng/uL) | uM
S502 | 57.62.47 | 3.7
S503 | 60.42.55 | 3.9
S505 | 93.92.43 | 6.0
N716 | 94.64.41 | 6.5
N718 | 88.64.98 | 6.1
N719 | 78.62.70 | 5.4

==================
Update 2/11/17

I compared pricing, assuming that the concentration of the indexes that Illumina provides is 5uM.

iDT Ultramers, 20nmole of each index in a 96-well plate was not significantly cheaper - within 10% of the cost of purchasing from Illumina. There doesn't appear to be a large cost savings here, so I'm opting to stick with Illumina indexes, which I'll aliquot into strip tubes.

Last edited by JVGen; 02-11-2017 at 08:45 AM.
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Old 07-17-2017, 05:48 PM   #2
georgeyeh
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Default How to calculate ssDNA concentration?

Could you quickly go over how you calculate the concentration based on Qubit ssDNA readouts?

Thank you!
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Old 07-17-2017, 07:09 PM   #3
JVGen
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Default

Quote:
Originally Posted by georgeyeh View Post
Could you quickly go over how you calculate the concentration based on Qubit ssDNA readouts?

Thank you!
I just took the index sequence and converted to moles/volume (molarity). Dust off your old pal Avogadro.
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