[Dominik]

Hello there,

I was wondering why it is 2 EtOH washes for the TNA protocols followed by 5-10 minutes of drying and 4 (or 2×4) EtOH washes followed by 30 minutes of drying.

What am I missing here?

best Dominik

[admin]

Ahoy,

Thank you for your interest.

This can be attributed to the individual protocols not having been created at exactly the same time and researcher, resulting in minor adjustments between them.

As to what is the better procedure.

The EtOH wash step is mostly aimed at washing off residual salts. Predominantly this is achieved through the 20% water but the 80% EtOH (70% would still work)  is needed to ensure that the TNA remains precipitated and bound to the beads.

Some samples show a higher tendency to carry along salts and some downstream applications are more susceptible to them.

You will see that whenever someone in the forum asks about an impurity shoulder in the absorption spectrum of the final extract we recommend  additional EtOH wash steps as the first possible solution. As long as the EtOH percentage remains high enough this should lead to little to no reduction in yield.

The gist, there is no clear answer.
More EtOH washes lead to a potentially cleaner extract but also take more time/material and may be unnecessary.

Best Regards

Tim M

 

[Dominik]

Hi Tim,

thanks for the quick reply. For DNA Extraction I usually use 2 washes of 80% EtOH and did not run into trouble with my PCR’s so I guess this is sufficient. For the TNA extraction I will go with 4 then, I will automatize the whole protocol anyways, then there is basically no difference between 2 and 4 washes except maybe a minute longer runtime.

[Author]

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