Quant 3D Q150 Guide de l'utilisateur télécharger pdf (Page 36)

Prepare Samples and Load

Reactions

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Prepare the DNA samples ............................................. 36

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Prepare the digital PCR reactions ....................................... 38

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Load the Digital PCR 20K Chips ........................................ 40

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Load the chips using the Chip Loader .................................. 42

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Load chips manually ................................................. 50

Prepare the DNA samples

We recommend the following best practices for the preparation of DNA template,

genomic DNA (gDNA) or complementary DNA (cDNA), for use in digital PCR

(dPCR) experiments. Because dPCR experiment strategy and methodology can vary

significantly, sample preparation and template quality must be assessed on an

individual basis.

To ensure high quality data, we recommend that the gDNA or cDNA template you

use for dPCR experiments:

• Is extracted from the raw material that you are testing with an optimized

protocol; salting-out procedures and crude lysates are not recommended

• Does not contain PCR inhibitors

• Has an A

260/230

and A

260/280

ratio between 1.7 and 1.9

Note: The ratio of absorbance at 260 nm and 280 nm is used to assess the purity

of DNA and RNA. A ratio of ~1.8 is generally accepted as “pure” for DNA; a ratio

of ~2.0 is generally accepted as “pure” for RNA. If the ratio is appreciably lower

in either case, it may indicate the presence of protein, phenol, or other

contaminants that absorb strongly at or near 280 nm.

The ratio of absorbance at 260 nm and 230 nm is used as a secondary measure of

nucleic acid purity. The 260/230 values for “pure” nucleic acid are often higher

than the respective 260/280 values. Expected 260/230 values are commonly in the

range of 2.0-2.2. If the ratio is appreciably lower than expected, it may indicate

the presence of contaminants that absorb at 230 nm.

The quantity of DNA template added to a dPCR reaction depends on the:

• Concentration of gDNA or cDNA present in each sample

• Expected number of copies of the target sequence present in the genome or

cDNA of your samples

Quality of DNA

Quantity of DNA

QuantStudio

3D Digital PCR System User Guide

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