Qubit dsDNA Broad Range Assay: Difference between revisions
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PreDec2022>Eichen (Created page with "__FORCETOC__ ==Goal:== Quantify DNA samples that will be sent out for sequencing. Qubit Assays produce more accurate and precise results than measurement by NanoDrop spectrophotometer. However, the materials required for the Qubit Assay are limited. Thus, we only perform this assay on samples slated for sequencing (which must match the concentration requirements described by the sequencing companies). ==Materials:== Qubit Fluorometer Invitrogen Qubit dsDNA Broad...") |
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__FORCETOC__ | __FORCETOC__ | ||
==Goal | ==Goal== | ||
Quantify DNA samples that will be sent out for sequencing. | Quantify DNA samples that will be sent out for sequencing. | ||
Qubit Assays produce more accurate and precise results than measurement by NanoDrop spectrophotometer. However, the materials required for the Qubit Assay are limited. Thus, we only perform this assay on samples slated for sequencing (which must match the concentration requirements described by the sequencing companies). | Qubit Assays produce more accurate and precise results than measurement by NanoDrop spectrophotometer. However, the materials required for the Qubit Assay are limited. Thus, we '''only perform this assay on samples slated for sequencing''' (which must match the concentration requirements described by the sequencing companies). | ||
==Materials== | |||
*Qubit Fluorometer | |||
*Qubit dsDNA Broad Range (BR) Assay Kit (Catalog #Q32850) | |||
**Buffer & Reagent / Dye | |||
**2 standards | |||
*Qubit Assay Tubes, 500ct. (Catalog #Q32856) | |||
'''Location & Storage:''' | |||
*<u>Fluorometer:</u> on the bench next to the plate imager | |||
*<u>Buffer & Reagent:</u> in a paper box inside the plant cabinet. Both must be stored at room temperature; reagent must be stored in the dark. | |||
*<u>Standards:</u> in a plastic Invitrogen Qubit BR Assay bag in the 4C fridge (behind the chemicals box containing Ethidium Bromide, etc). Must be stored at 4C? | |||
*<u>Qubit Assay Tubes:</u> autoclaved; in tinfoil-covered glass beakers inside the plant cabinet. | |||
Fluorometer: on the bench next to the plate imager | |||
Buffer & Reagent: in a paper box inside the plant cabinet. Both must be stored at room temperature; reagent must be stored in the dark. | |||
Standards: in a plastic Invitrogen Qubit BR Assay bag in the 4C fridge (behind the chemicals box containing Ethidium Bromide, etc). Must be stored at 4C? | |||
Qubit Assay Tubes: autoclaved; in tinfoil-covered glass beakers inside the plant cabinet. | |||
==Assay Setup== | |||
Based on the number of samples you wish to quantify, determine master mix (buffer + reagent) volume. | |||
Use the Qubit Fluorometer’s '''Reagent Calculator''' (located at the bottom of the screen display) to calculate volume: | |||
*Enter the number of samples | |||
*Always include 2 standards, unless you’ve already run standards that same day | |||
*Include “overage” (to ensure there is enough for loading) | |||
Set up & label Qubit Assay tubes for your samples + 2 standards. | Set up & label Qubit Assay tubes for your samples + 2 standards. | ||
Make the master mix. | Make the master mix. | ||
Depending on the volume required, I use either a 1.5ml microcentrifuge tube or a 15ml falcon tube | *Depending on the volume required, I use either a 1.5ml microcentrifuge tube or a 15ml falcon tube | ||
Vortex briefly to mix | *Vortex briefly to mix | ||
Pipette the | <u>Pipette the master mix into the assay tubes:</u> | ||
*190ul / standard tube | |||
*198ul / sample tube | |||
<u>Pipette the DNA into the correct assay tubes:</u> | |||
*10ul / standard tube (Standard #1 & Standard #2) | |||
*2ul / sample tube | |||
Vortex each sample for 2-3 seconds & incubate at room temperature for 2 minutes. | |||
==Qubit Fluorometer Quantification | ==Qubit Fluorometer Quantification== | ||
From the Home screen, choose dsDNA assay, then Broad Range. Select “Run Standards”, then load Standard #1 & measure. Repeat for Standard #2. | From the Home screen, choose dsDNA assay, then Broad Range. Select “Run Standards”, then load Standard #1 & measure. Repeat for Standard #2. | ||
Next, measure your samples. Enter the correct volume of sample added to the assay tube (usually 2ul), then load & quantify samples one by one. | Next, measure your samples. Enter the correct volume of sample added to the assay tube (usually 2ul), then load & quantify samples one by one. | ||
==Cleanup | ==Cleanup== | ||
Dispose of the used assay tubes & unplug the Qubit fluorometer. | Dispose of the used assay tubes & unplug the Qubit fluorometer. | ||
Latest revision as of 12:57, 16 December 2022
Goal
Quantify DNA samples that will be sent out for sequencing.
Qubit Assays produce more accurate and precise results than measurement by NanoDrop spectrophotometer. However, the materials required for the Qubit Assay are limited. Thus, we only perform this assay on samples slated for sequencing (which must match the concentration requirements described by the sequencing companies).
Materials
- Qubit Fluorometer
- Qubit dsDNA Broad Range (BR) Assay Kit (Catalog #Q32850)
- Buffer & Reagent / Dye
- 2 standards
- Qubit Assay Tubes, 500ct. (Catalog #Q32856)
Location & Storage:
- Fluorometer: on the bench next to the plate imager
- Buffer & Reagent: in a paper box inside the plant cabinet. Both must be stored at room temperature; reagent must be stored in the dark.
- Standards: in a plastic Invitrogen Qubit BR Assay bag in the 4C fridge (behind the chemicals box containing Ethidium Bromide, etc). Must be stored at 4C?
- Qubit Assay Tubes: autoclaved; in tinfoil-covered glass beakers inside the plant cabinet.
Assay Setup
Based on the number of samples you wish to quantify, determine master mix (buffer + reagent) volume. Use the Qubit Fluorometer’s Reagent Calculator (located at the bottom of the screen display) to calculate volume:
- Enter the number of samples
- Always include 2 standards, unless you’ve already run standards that same day
- Include “overage” (to ensure there is enough for loading)
Set up & label Qubit Assay tubes for your samples + 2 standards.
Make the master mix.
- Depending on the volume required, I use either a 1.5ml microcentrifuge tube or a 15ml falcon tube
- Vortex briefly to mix
Pipette the master mix into the assay tubes:
- 190ul / standard tube
- 198ul / sample tube
Pipette the DNA into the correct assay tubes:
- 10ul / standard tube (Standard #1 & Standard #2)
- 2ul / sample tube
Vortex each sample for 2-3 seconds & incubate at room temperature for 2 minutes.
Qubit Fluorometer Quantification
From the Home screen, choose dsDNA assay, then Broad Range. Select “Run Standards”, then load Standard #1 & measure. Repeat for Standard #2.
Next, measure your samples. Enter the correct volume of sample added to the assay tube (usually 2ul), then load & quantify samples one by one.
Cleanup
Dispose of the used assay tubes & unplug the Qubit fluorometer.