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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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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. -
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!Comment
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High temperature can also impact AT rich sequences and degrade them.Comment
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Interesting. I'll look into this a bit more, thanks.Originally posted by huguesparri View PostComment
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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.Comment
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...
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