Microplate Assays for Nucleic Acids

Accurate, efficient nucleic acid quantitation is critical for molecular biology labs that work with precious, rare, or difficult-to-process samples and applications requiring precise measurement. These include real-time PCR, measuring yields of RNA preparations for microarray experiments, reverse transcription PCR (RT-PCR), differential display PCR, northern blot analysis, S1 nuclease assays, RNase protection assays, and cDNA library preparations.

dsDNA assays

PicoGreen® reagent assays provide a rapid and simple fluorescence-based method for the accurate quantitation of dsDNA. The assays are many times more sensitive than conventional UV absorbance and can be optimized for different concentration ranges. PicoGreen® reagent assays are routinely used for genotyping by allele-specific PCR, quantitating dsDNA samples before and after PCR amplification, and determining PCR amplification yields before sequencing.

ssDNA assays

OliGreen® reagent assays provide a rapid and simple fluorescence-based method for the accurate quantitation of ssDNA. The assay is many times more sensitive than conventional UV absorbance, and free nucleotides or short oligonucleotides of up to 6 bases do not interfere with the OliGreen® ssDNA quantitation assay. OliGreen® reagent assays are routinely used for antisense oligonucleotides, aptamers, genomic DNA isolated under denaturing conditions, PCR primers, sequencing primers, and single-stranded phage DNA.

RNA assays

RiboGreen® reagent assays provide a rapid and simple fluorescence-based method for the accurate quantitation of RNA. RiboGreen® reagent does not detect significant sample contamination by free nucleotides and thus more accurately measures the amount of intact RNA in potentially degraded samples. The RiboGreen® reagent is ideal for fast and accurate measurement of RNA before generating cDNA, northern blot analysis, S1 nuclease assays, RNase protection assays, reverse transcription PCR, and differential display PCR.

     

PicoGreen® dsDNA Quantitation Reagent
Linear quantitation of calf thymus DNA from 25 pg/mL to 1,000 ng/mL using PicoGreen® dsDNA Quantitation Reagent. Samples were excited at 480 nm, and the fluorescence emission intensity was measured at 520 nm and plotted as a function of DNA concentration. The inset shows the results obtained with DNA concentrations between 0 and 750 pg/mL.

Selection guides
Name Quant-iT™ PicoGreen® dsDNA Assay Kit Quant-iT™ DNA Assay Kit, high sensitivity Quant-iT™ dsDNA Assay Kit, broad range
Target dsDNA dsDNA dsDNA
Reporter PicoGreen® PicoGreen® PicoGreen®
Ex/Em (nm) 502/523 502/523 502/523
Sample Selectively quantify dsDNA in the presence of ssDNA, RNA, or free nucleotides Selectively quantify dsDNA in the presence of ssDNA, RNA, or free nucleotides Selectively quantify dsDNA in the presence of ssDNA, RNA, or free nucleotides
Usage Lower detection limit 250 pg/mL dsDNA (50 pg in a 200 µL assay volume) Lower detection limit 0.2–100 ng of DNA in 200 µL assay volume Detects in the range of 2–1,000 ng of DNA in 200 µL assay volume
Components Includes buffer and DNA standard Includes buffer and DNA standard Includes buffer and DNA standard
Format 2,000 assays at 200 µL per assay 1,000 assays, with a 200 µL assay volume in a 96-well microplate format 1,000 assays, with a 200 µL assay volume in a 96-well microplate format
Protocol outline
  1. Dilute samples in microplate wells.
  2. Add working solution of PicoGreen® reagent.
  3. Incubate at room temperature for 2–5 min.
  4. Measure fluorescence.
  5. Quantitate the dsDNA by comparing to standard curve.
Cat. No. P11496 Q33120 Q33130
  Quant-iT™ OliGreen® ssDNA Reagent Quant-iT™ OliGreen® ssDNA Assay Kit
Target ssDNA ssDNA
Reporter OliGreen® OliGreen®
Ex/Em (nm) 500/525 500/525
Sample Selectively quantify oligonucleotides and single-stranded DNA (ssDNA) in solution Selectively quantify oligonucleotides and single-stranded DNA (ssDNA) in solution
Usage Detect 200 pg to 2 µg ssDNA in a 200 µL assay volume Detect 200 pg to 2 µg ssDNA in a 200 µL assay volume
Components Bulk reagent Includes buffer and DNA standard
Assay format 2,000 assays using a 200 µL assay volume 2,000 assays using a 200 µL assay volume
Protocol outline
  1. Dilute samples in microplate wells.
  2. Add working solution of the OliGreen® reagent.
  3. Incubate at room temperature for 2–5 min.
  4. Measure fluorescence.
  5. Quantitate the ssDNA by comparing to standard curve.
Cat. No. O7582 O11492
  Quant-iT™ RiboGreen® RNA Assay Kit RediPlate™ 96 RiboGreen® RNA Quantitation Kit
Target RNA RNA
Reporter RiboGreen® RiboGreen®
Ex/Em (nm) 500/525 500/525
Assay Selectively quantify RNA in solution Selectively quantify RNA in solution
Usage Detection range 1–200 ng RNA Detection range 3–200 ng RNA
Components Kit includes reagent, buffers, and RNA standard Pre-dispensed in 96-well plate
Format 2,000–20,000 microplate assays using a 200 µL sample volume 96 assays
Protocol outline
  1. Dilute samples in microplate wells.
  2. Add RiboGreen® reagent working solution.
  3. Incubate at room temperature for 2–5 min.
  4. Measure fluorescence.
  5. Quantitate the RNA by comparing to standard curve.
Cat. No. R11490 R32700
dsDNA assays
Name Quant-iT™ PicoGreen® dsDNA Assay Kit Quant-iT™ DNA Assay Kit, high sensitivity Quant-iT™ dsDNA Assay Kit, broad range
Target dsDNA dsDNA dsDNA
Reporter PicoGreen® PicoGreen® PicoGreen®
Ex/Em (nm) 502/523 502/523 502/523
Sample Selectively quantify dsDNA in the presence of ssDNA, RNA, or free nucleotides Selectively quantify dsDNA in the presence of ssDNA, RNA, or free nucleotides Selectively quantify dsDNA in the presence of ssDNA, RNA, or free nucleotides
Usage Lower detection limit 250 pg/mL dsDNA (50 pg in a 200 µL assay volume) Lower detection limit 0.2–100 ng of DNA in 200 µL assay volume Detects in the range of 2–1,000 ng of DNA in 200 µL assay volume
Components Includes buffer and DNA standard Includes buffer and DNA standard Includes buffer and DNA standard
Format 2,000 assays at 200 µL per assay 1,000 assays, with a 200 µL assay volume in a 96-well microplate format 1,000 assays, with a 200 µL assay volume in a 96-well microplate format
Protocol outline
  1. Dilute samples in microplate wells.
  2. Add working solution of PicoGreen® reagent.
  3. Incubate at room temperature for 2–5 min.
  4. Measure fluorescence.
  5. Quantitate the dsDNA by comparing to standard curve.
Cat. No. P11496 Q33120 Q33130
ssDNA assays
  Quant-iT™ OliGreen® ssDNA Reagent Quant-iT™ OliGreen® ssDNA Assay Kit
Target ssDNA ssDNA
Reporter OliGreen® OliGreen®
Ex/Em (nm) 500/525 500/525
Sample Selectively quantify oligonucleotides and single-stranded DNA (ssDNA) in solution Selectively quantify oligonucleotides and single-stranded DNA (ssDNA) in solution
Usage Detect 200 pg to 2 µg ssDNA in a 200 µL assay volume Detect 200 pg to 2 µg ssDNA in a 200 µL assay volume
Components Bulk reagent Includes buffer and DNA standard
Assay format 2,000 assays using a 200 µL assay volume 2,000 assays using a 200 µL assay volume
Protocol outline
  1. Dilute samples in microplate wells.
  2. Add working solution of the OliGreen® reagent.
  3. Incubate at room temperature for 2–5 min.
  4. Measure fluorescence.
  5. Quantitate the ssDNA by comparing to standard curve.
Cat. No. O7582 O11492
RNA assays
  Quant-iT™ RiboGreen® RNA Assay Kit RediPlate™ 96 RiboGreen® RNA Quantitation Kit
Target RNA RNA
Reporter RiboGreen® RiboGreen®
Ex/Em (nm) 500/525 500/525
Assay Selectively quantify RNA in solution Selectively quantify RNA in solution
Usage Detection range 1–200 ng RNA Detection range 3–200 ng RNA
Components Kit includes reagent, buffers, and RNA standard Pre-dispensed in 96-well plate
Format 2,000–20,000 microplate assays using a 200 µL sample volume 96 assays
Protocol outline
  1. Dilute samples in microplate wells.
  2. Add RiboGreen® reagent working solution.
  3. Incubate at room temperature for 2–5 min.
  4. Measure fluorescence.
  5. Quantitate the RNA by comparing to standard curve.
Cat. No. R11490 R32700
Thermo Scientific plate readers

Expertly detect fluorescence with Thermo Scientific plate readers

High-sensitivity fluorescence detection for 96-1,536 samples can be quickly performed on Fluoroskan or Fluoroskan FL Microplate Fluorometer or Varioskan LUX Multimode Microplate Reader using Invitrogen reagents for optimal detection. Take advantage of automatic dynamic range selection to get optimal gain settings for each individual well and automation capabilities for even higher throughput.

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Resources

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