UK Technical Service Team FAQs

Our Technical Service team plays a vital role in assisting customers with a wide range of technical, product, and instrumentation questions. In 2024, they tackled numerous inquiries, addressing everything from Maxwell Elution Buffers to Microplate Recommendations.

1. What are Maxwell Elution Buffers? 

Maxwell Elution Buffers are tailored solutions for nucleic acid extraction using the Maxwell system. Designed for optimal performance, they ensure excellent yield, purity, and stability.

2. What types are available?

Here's a quick guide to the different Maxwell Elution Buffers and their compositions: 

Part Number	Buffer Name	Composition
A828	Elution Buffer (DNA IQ)	10 mM Tris, 0.1 mM EDTA, pH 8.0
MC112	Elution Buffer	Same as A828; 10 mM Tris, 0.1 mM EDTA, pH 8.0
MC139	Elution Buffer	10 mM Tris, 0.1 mM EDTA, pH 9.0
MC1511	PCR Elution Buffer (EBE)	10 mM Tris, pH 9.0
MC1521	NGS Elution Buffer (EBF)	10 mM Tris, 10 mM NaCl, pH 9.0
MD142	Elution Buffer (DNA)	10 mM Tris, 0.1 mM EDTA, pH 8.5
V852	MagneHis� Elution Buffer	500 mM imidazole, 100 mM HEPES, pH 7.5 (with NaOH)

3. How do I choose the right buffer? 

The buffer you need depends on the step in the nucleic acid extraction protocol and the type of sample you're working with. Each kit comes with an optimised buffer. Refer to the table above to understand the specific compositions of the elution buffer contained in your specific kit. This information can be found on the product webpage or in the Technical Manual. 

4. Can I substitute Maxwell buffers? 

The composition of each elution buffer is critical for successful elution of nucleic acids. Buffered solutions, such as those containing Tris, help maintain a stable pH environment, which is essential for preserving the integrity of nucleic acids during elution and storage. This stability helps prevent nucleic acid degradation, ensuring the samples remain viable for downstream applications.

1. What are the common methods for DNA quantitation?

• Absorbance (Spectrophotometry)
• Fluorescence
• Agarose Gel Electrophoresis
• Quantitative PCR (qPCR)

2. What are the advantages and disadvantages of absorbance? 

• Advantages: Simple, rapid analysis with no additional reagents, provides purity information.
• Disadvantages: Overestimates due to contaminants; not useful at low nucleic acid concentrations.

Spectrophotometry can overestimate nucleic acid quantity because it measures absorbance at 260 nm, which is not exclusive to DNA or RNA. Other substances, such as proteins, free nucleotides, phenol, and other organic compounds, also absorb at this wavelength, leading to higher readings. Additionally, contaminants like RNA and residual phenol from extraction processes can contribute to the absorbance, resulting in an overestimation of the actual nucleic acid concentration. This method lacks the specificity needed to distinguish between DNA, RNA, and other absorbing compounds.

3. What are the advantages and disadvantages of fluorescence? 

• Advantages: Highly sensitive, nucleic acid-specific.
• Disadvantages: Requires dyes, has photobleaching risks, and requires a plate reader or fluorometer.

Fluorescent DNA-binding dyes work by intercalating between the base pairs of double-stranded DNA or binding to the grooves of DNA. When the dye binds to DNA, its fluorescence increases significantly, allowing for the detection and quantification of DNA. The intensity of the fluorescence is proportional to the amount of DNA present. These dyes are specific to nucleic acids, which makes them useful for precise DNA quantitation. Some common fluorescent DNA-binding dyes include Promega’s QuantiFluor® dsDNA Dye and Ethidium Bromide. These can be easily processed in higher throughput using a plate reader.

4. What are the advantages and disadvantages of agarose gel electrophoresis? 

• Advantages: Estimates size and quantity, distinguishes contaminants.
• Disadvantages: Less precise, time-consuming.

Running nucleic acid samples on a gel is not considered quantitative because it primarily provides a visual estimation rather than precise measurements. The intensity of the bands on the gel can give a rough idea of the DNA or RNA concentration, but this method lacks the sensitivity and accuracy required for precise quantification. Variability in gel loading, staining efficiency, and the subjective nature of visual assessment contribute to its limitations in providing accurate, reproducible quantitation.

5. What are the advantages and disadvantages of quantitative PCR (qPCR)? 

• Advantages: Extremely sensitive, allows precise quantitation even in complex mixtures.
• Disadvantages: Requires specialised equipment, expensive reagents, and is prone to contamination leading to false positives.

PCR, particularly quantitative PCR (qPCR), is considered the gold standard for nucleic acid quantification due to its high sensitivity, specificity, and precision. Unlike other methods, qPCR can detect and quantify even minute amounts of DNA or RNA with great accuracy. It amplifies the target nucleic acid, allowing for the detection of low-copy-number targets in complex samples. This method also offers real-time monitoring of the amplification process, providing precise quantitation across a wide dynamic range. Additionally, qPCR is less influenced by contaminants that affect other quantification methods, ensuring reliable results.

For more detailed information, you can visit Promega's resource on DNA quantitation. 

 

1. How to choose the right plate? 

• For optimal results, we recommend using white plates for luminescent assays and black plates for fluorescence assays.
• In most cases, we’ve found the microplate brand itself bears little weight on the assay results (assuming the customer is performing the assay correctly and has set up their microplate reader correctly).
• For customers multiplexing with our luminescent and fluorescent assays, the use of either white or black plates is acceptable.  The customer can expect to observe ~10-to 15-fold reduction in RLU signal when using a black plate for a luminescent assay.
• Cell-based assays require plates that are surface-treated as ‘Tissue Culture Treated’.

2. What plates do you recommend? 

Below we’ve provided some examples of plates we recommend. Please note there are many good microplate vendors that can be recommended to our customers. Give Technical Support a call to find out more.

Plates for Biochemical Assays: 96-well white plates suitable for luminescent biochemical assays (no cells)

• Corning | 96 Wells White
• Greiner Bio-One | 96 Wells White
• Nunc | 96 Wells White

Plates for Biochemical Assays: 96-well black plates suitable for fluorescent biochemical assays (no cells)

• Azenta Life Sciences | 96 Wells Black
• Corning | 96 Wells Black
• Greiner Bio-One | 96 Wells Black

Plates for Cell-Based Assays: 96-well white TC plates suitable for luminescent cell-based assays

• Corning | 96 Wells White
• Greiner Bio-One | 96 Wells White

Plates for Cell-Based Assays: 96-well black TC plates suitable for fluorescent cell-based assays

• Corning | 96 Black Wells
• Greiner Bio-One | 96 Black Wells

Plates for Determining Protein and/or Nucleic Acid Concentrations: 96-well UV Transparent Plates

• Corning | 96 Wells Clear, UV Transparent
• Corning | Half Area 96 Wells Clear
• Greiner Bio-One | UV-Star Microplate

1. What wavelength do I need to set for my Luminometer that uses Photomultiplier Tubes (PMTs)? 

Luminometers, such as Promega’s GloMax range, utilise photomultiplier tubes (PMTs), which are highly sensitive detectors capable of measuring luminescence across a broad range of wavelengths, typically from 300 nm to 900 nm with a focus on the visible range (400-700 nm). Since PMTs detect photons across this entire range, it is generally not necessary to define a specific peak wavelength for luminescent reporter assays when using these instruments. The exception would be when carrying out a BRET type assay such as NanoBRET®.

2. What if my Luminometer Requires Specific Wavelengths? 

In contrast, some luminometers equipped with monochromators require the user to set a specific wavelength, to measure the luminescent signal accurately. For these instruments the following peak emission wavelengths are recommended:

• Firefly luciferase: Peak emission at around 560 nm (green light)
• Renilla luciferase: Peak emission at around 480 nm (blue light)
• NanoLuc luciferase: Peak emission at around 460 nm (blue light)

If you’d like more detailed or specific advice about anything in this article, reach out to the Technical Service Team.

Email: uktechserve@promega.com

Phone : (+44) 23 8071 7371[MC1] or 0800 559 900

Hours: Monday – Thursday, 8:30 am – 5:30 pm and Friday, 8:30 am – 4:00 pm