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Old 07-10-2013, 08:07 AM   #6
Calluna
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Location: Seattle, WA

Join Date: Feb 2012
Posts: 12
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Call Illumina and ask if 0.1N NaOH will be strong enough.

If not, you can use a higher concentration NaOH, but less of it... and then neutralize.

I think you can do the following (but double check my math!)
Denature:
6.5uL total of your 1.1nm normalized/mixed libraries
2uL of 0.5N NaOH
1.5uL H2O
Total denature volume = 10uL

Add 1uL of 1N HCl
Total neutralized denature = 11 uL

Add 639uL HT1

Total final volume = 650uL

If I did the math correctly, I think that gives you an 11pM final concentration, and because you neutralized the NaOH, you shouldn't have to worry about pH.
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