Oct 22, 2020

Public workspaceIllumina TruSeq Library quantification with qPCR probe method

This protocol is a draft, published without a DOI.
Illumina TruSeq Library quantification with qPCR probe method
  • Kentaro Itokawa1
  • 1National Institute of Infectious Diseases, Japan
Kentaro Itokawa
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Protocol Citation: Kentaro Itokawa 2020. Illumina TruSeq Library quantification with qPCR probe method. protocols.io https://protocols.io/view/illumina-truseq-library-quantification-with-qpcr-p-bnpamdie
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 21, 2020
Last Modified: October 22, 2020
Protocol Integer ID: 43458
Keywords: truseq type adapter, quantification of illumina library, qpcr probe method, qpcr, illumina library, size distribution of library, probe, library, illumina
Abstract
A homemade solution for quantification of Illumina library.

Because the method uses a quenched-probe (not intercalator), it is expected to be relatively stable for the difference of size distribution of libraries.

Currently, we confirmed this protocol with a probe for TruSeq type adapter.
Materials
MATERIALS
ReagentUltraPure™ Salmon Sperm DNA SolutionThermo FisherCatalog #15632011
ReagentPhiX Control v3Illumina, Inc.Catalog #FC-110-3001
ReagentPrimeTime® Gene Expression Master MixIntegrated DNA Technologies, Inc. (IDT)Catalog #1055770
Troubleshooting
Preparing calibrators and primer/probe
Prepare low-TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) containing 5 ng/μl salmon sperm DNA.
1 M Tris-HCl, pH8.0100 μl
0.5 M EDTA10 μl
UltraPure™DNA Solution 10 mg/mL5 μl
milli-Q water9889 μl
Total10 mL

Store in -20 °C.
Prepare dilution series of PhiX control with low-TE buffer containing 5 ng/μl salmon sperm DNA in 8-strip PCR tubes.
These are used as calibrators.
50 pM100 μl
5 pM100 μl
0.5 pM100 μl
0.05 pM100 μl

Store in -20 °C.
Prepare 20x primers & probe mix solution as below.

Probe5 μM
P5 primer5 μM
P7 primer5 μM

Store in -20 °C.

Double-quenched probe (IDT): /56-FAM/ACACTCTTT/ZEN/CCCTACACGACGCTCTTC/3IABkFQ/

P5 primer: 5'-AATGATACGGCGACCACCGA-3'
P7 primer: 5'-CAAGCAGAAGACGGCATACGA-3’
Prepare the following qPCR master mix per reactions for the number of your samples (+ calibrators).

Prime Time PCR master mix10 μl
20x primers & probe mix1 μl
H2O8 μl

Distribute above to each well.

Add 1 μl of template (diluted library or calibrator) to each well.
Conduct PCR with the following condition.

95 °C for 2 min
30 cycles of
97 °C for 5s
62 °C for 10s
68 °C for 15s (photo with a filter appropriate for SYBR-green or FAM)
Result

An example of expected result
Calculate the original concentration of your library.