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Old 07-17-2013, 09:13 AM   #9
Calluna
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Location: Seattle, WA

Join Date: Feb 2012
Posts: 12
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I'm not sure how crucial it is to have your denature NaOH concentration at at least 0.1N, so I wouldn't mess with it if you don't have to.

This is what I would suggest instead:

Denature:
5uL of 1.1nM sample A
5uL of 1.1nM sample B
2.5uL of 0.5N NaOH (or 1.25uL H2O + 1.25uL 1N NaOH...either way it is the same thing. This way, you maintain a final effective NaOH concentration in the denature if 0.1N)
Denature volume = 12.5uL

Neutralize:
add 1.25uL of 1N HCl
Neutralized volume = 13.75uL

Hyb mix:
add 986.25uL HT1
Total final volume of 1000uL.

Don't forget to use some PhiX to help with any diversity issues.
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