Agilent eBook - 24

Best Practices with an Agilent Parallel CE System

Additionally, storage temperature should be taken
into consideration. If you are handling high molecular
weight DNA it is important that you minimize the
number of freeze/thaw cycles as this can contribute to
faster degradation. We recommend customers aliquot
their high molecular weight gDNA prior to storage.
Regardless of sample type, it is important that you
maintain aseptic techniques so that you are not
introducing any RNases or DNases into your sample.
Second, it is important that you handle the quality control
kit reagents properly. Mishandling of kit reagents can
lead to degradation of kit components which will
adversely affect the quality control analysis. Handling of
kit components according to the manufacturer's guidelines ensures that the results from the quality control
analysis are accurate representations of your sample.
Finally, how you mix your sample prior to quality
control analysis can also drastically influence the
results. It is important to mix both your sample and
the manufactured supplied reagents according to
manufacturer's instructions prior to use. If the provided
reagents are to be mixed with your sample, it is
important that this is done to manufacturer's
specifications. This ensures that when either you or
the quality control instrument is sampling an aliquot,
that aliquot is representative of the entire sample.
Without proper mixing, you can get improper sampling,
which will lead to inconsistent results.



GEN: The quality of genomic DNA can vary widely.
How do different extraction methods impact quality
and DNA size?

extracting the gDNA. Utilizing QC steps ensures
that your extracted material is suitable for your

KL: Different kits and protocols can drastically
influence the size of the extracted gDNA. Depending
on your application, different initial gDNA sizes are
required. For example, a long-read sequencing
application requires significantly higher molecular
weight gDNA than short-read sequencing. By screening
your extracted gDNA you ensure that the kit/protocol
used is supplying gDNA that is appropriate for your
application. We have screened several commercially
available kits here at Agilent and have found that they
can vary widely in the size of gDNA extracted, even with
samples from the same source. Surprisingly, a phenol
chloroform extraction consistently provides the highest
molecular weight gDNA compared to any other
extraction method that we have tried.

Regardless of the extraction method used and the
sample type you are extracting from, how you handle
the extracted gDNA can drastically influence your
results. For instance, high molecular weight DNA, if
not handled carefully and properly, can be sheared
simply by pipetting. We strongly recommend our
customers use wide-bore pipet tips when handling
high molecular weight gDNA. Since the average gDNA
size can impact library preparation protocols, in
particular shearing times, we recommend performing
quality control analysis after storage and prior to
library prep. Looking at the quality of the gDNA
immediately before library preparation ensures that
you know the quality of your sample, and alleviates
concern that the sample may have changed during
storage. Proper quality control of samples ultimately
gives you confidence in your sample's suitability for
downstream applications. n

Different tissue types can also impact the extracted
gDNA size distribution. There are certain cancer cell
lines that consistently give higher-quality, higher
molecular weight extractions than others. Why this
is the case is still an open question. It is important to
understand that even if you are using a kit or method
that you expect will provide the highest molecular
weight DNA possible, that you still may end up with
lower molecular weight DNA simply due to the sample
type and how that sample was handled prior to you
For Research Use Only. Not for use in diagnostic procedures.
This information is subject to change without notice.
© Agilent Technologies, Inc. 2019

Agilent eBook

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