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Old 09-08-2019, 10:16 PM   #1
Carcharodon
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Default Why is it commonly recommended to Speed-Vac without heat?

Especially when it comes to Illumina libraries, why is it commonly recommended to Speed-Vac without heat? Is this to prevent (or slow down) potential DNase activity?
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Old 09-09-2019, 03:37 PM   #2
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I believe this recommendation usually applies to RNA samples - which are more heat sensitive.

Depending on the buffer (some labs might use be pure H2O) and the lengths of fragments, using low temperatures (e.g. room temperature) might avoid denaturation of DNA samples.
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Old 09-09-2019, 03:55 PM   #3
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Thanks for the response, luc! I'm currently working through a ddRAD protocol (my second library). Long story short I have digested DNA w/ ligated adapters. I bead-cleaned them after pooling, but eluted with a large volume of HPLC H2O and Speed-Vacced to reduce the volume (from ~ 150 uL to ~ 30 uL).

Our normal vacuum pump is broken so I'm using a weaker replacement. It was taking forever so I upped the temp to ~ 45 C.

...and then, as often happens, I made myself paranoid. But I'm also genuinely curious about this recommendation. So thanks!
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Old 09-10-2019, 01:15 AM   #4
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High temperature can also impact AT rich sequences and degrade them.
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Old 09-10-2019, 10:22 PM   #5
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Quote:
Originally Posted by huguesparri View Post
High temperature can also impact AT rich sequences and degrade them.
Interesting. I'll look into this a bit more, thanks.
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Old 09-11-2019, 04:14 PM   #6
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Very, very likely your libraries will be perfectly fine. 45C should not be a problem, even in pure H2O. This is my guess, though.
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