Agilent TapeStation
All RNA and DNA quality control for library construction is done on an Agilent TapeStation 4150. This service is only offered as part of quantification for library preparation and sequencing performed by AGTC and is not offered as a stand-alone service.
RNA
| High Sensitivity RNA ScreenTape Assay | RNA ScreenTape Assay | |
|---|---|---|
| sample volume required | 2 μL | 1 μL |
| sizing range | 25 - 6000 bp | 25 - 6000 bp |
| sensitivity | 0.1 ng/μL | 5 ng/μL |
| quantitative precision | 15% CV | 10% CV |
| quantitative accuracy | ±30% | ±20% |
| quantitative range | 0.5 - 10 ng/μL | 25 - 500 ng/μL |
| RINe functional range | 1 - 25 ng/μL | 25 - 500 ng/μL |
| maximum buffer concentration in sample | 10 mM Tris, 1 mM EDTA | 200 mM Tris, 20 mM EDTA, or 50 mM NaCl |
DNA
| High Sensitivity DNA 1000 ScreenTape Assay | DNA 1000 ScreenTape Assay | |
|---|---|---|
| sample volume required | 2 μL | 1 μL |
| sizing range | 35 - 1000 bp | 35 - 1000 bp |
| typical resolution | 35 - 300 bp: 15%, 300 - 1000 bp: 10%) | 35 - 300 bp: 15%, 300 - 1000 bp: 10% |
| sensitivity | 0.005 ng/μL | 0.1 ng/μL |
| sizing precision | 5% CV | 5% CV |
| sizing accuracy | ±10% (w/ electronic ladder: ±20%) | ±10% (w/ electronic ladder: ±20%) |
| quantitative precision | 15% CV | 0.1 - 1 ng/μL: 15% CV, 1 - 50 ng/μL: 10% CV |
| quantitative accuracy | ±20% | ±20% |
| quantitative range | 0.01 - 1 ng/μL | 0.1 - 50 ng/μL |
| maximum buffer concentration in sample | 7 mM KCl, 20 mM phosphate buffer, 20 mM guanidine-HCl, 80 mM NaCl, 20 mM C2H4O2-Na | 60 mM phosphate buffer, 20 mM KCl, 60 mM guanidine-HCl, 240 mM NaCl, 60 mM C2H4O2-Na |
Invitrogen Qubit
Qubit assays utilize fluorescent molecules that are usually highly selective for a particular nucleic acid (e.g. dsDNA over RNA, RNA over DNA) and accurate in the presence of DNA, salts, solvents, proteins, and free nucleotides. Prior to library construction, input RNA and DNA samples are quantified using the Qubit Fluorometer. Qubit fluorometric quantitation measures only the nucleic acid of interest, so the number is almost always lower than the A260 reading because UV absorbance readings measure anything that absorbs at 260 nm, including DNA, RNA, protein, free nucleotides, and excess salt. This service is only offered as part of quantification for library preparation and sequencing performed by AGTC and is not offered as a stand-alone service.
RNA
| RNA High Sensitivity Assay | RNA Broad Range Assay | |
|---|---|---|
| sample volume required | 1-20 μL | 1-20 μL |
| sample conc. range | 0.25 - 100 ng/μL | 1 - 1000 ng/μL |
| sample amount range | 4 - 200 ng | 10 - 1200 ng |
| assay conc. range (total) | 0.02 - 1 ng/μL | 0.05 - 6 ng/μL |
| assay conc. range (highest confidence) | 0.025 - 0.50 ng/μL | 0.10 - 5 ng/μL |
| maximum contaminant conc. in assay | 10 mM NaCl, 2 mM MgCl2, 10 mM C2H4O2-Na/C2H4O2-H3N 1% EtOH, 0.1% Phenol, 1X NTPs/oligos/ssDNA/dsDNA ... | 10 mM NaCl, 2 mM MgCl2, 10 mM C2H4O2-Na/C2H4O2-H3N 0.1% EtOH, 0.1% Phenol, 1X NTPs/oligos/ssDNA/dsDNA ... |
DNA
| 1X dsDNA High Sensitivity Assay | dsDNA Broad Range Assay | |
|---|---|---|
| sample volume required | 1-20 μL | 1-20 μL |
| sample conc. range | 0.01 - 100 ng/μL | 0.10 - 1000 ng/μL |
| sample amount range | 0.10 - 120 ng | 2 - 2000 ng |
| assay conc. range (total) | 0.0005 - 0.60 ng/μL | 0.01 - 10 ng/μL |
| assay conc. range (highest confidence) | 0.001 - 0.50 ng/μL | 0.01 - 5 ng/μL |
| maximum contaminant conc. in assay | 50 mM NaCl, 1 mM MgCl2, 30 mM C2H4O2-Na, 25 mM C2H4O2-H3N 1% EtOH, 0.1% Phenol, 1X dNTPs/ssDNA/RNA ... | 10 mM NaCl, 2 mM MgCl2, 10 mM C2H4O2-Na/C2H4O2-H3N 1% EtOH, 0.1% Phenol, 100 μM dNTPs, 6X RNA, 1X ssDNA, 3X oligos ... |
LightCycler 96
For some types of libraries, quantification prior to sequencing is done on the Roche LightCycler 96 to ensure equimolar pooling and appropriate loading concentrations.
Library Quantification by qPCR
Library quantification kits provide accurate and reliable absolute quantification of libraries prepared for sequencing on Illumina platforms. The kit functions across a wide range of library types, concentrations, fragment distributions and GC content.