MagMAX™ DNA Multi-Sample Ultra Kit (with extra proteinase K and DNA binding beads), 2,500 preps - FAQs

When using the PowerUp SYBR Green Master Mix (Cat. No. A25741, A25742, A25743, A25776, A25777, A25778, A25779, A25780, A25919), I get high Ct values. What can I do?

It could be due to the following reasons:

• Insufficient cDNA template is present. Use 10 to 100 ng of cDNA template per 50 µL reaction.
• Quality of cDNA template is poor. Quantify the amount of cDNA template and test the cDNA template for the presence of PCR inhibitors.
• Sample degradation. Prepare fresh cDNA, then repeat the experiment.
• Reduced number of PCR cycles in the thermal cycling protocol. Increase the number of PCR cycles to the default setting of 40.
• Primer-dimer formation and residual polymerase activity. Prepare the reaction mixes and reaction plate on ice. To ensure optimal results, run the reaction plate as soon as possible after completing the reaction setup.

When using the PowerUp SYBR Green Master Mix (Cat. No. A25741, A25742, A25743, A25776, A25777, A25778, A25779, A25780, A25919), the Multicomponent signal for ROX is not linear. What could cause this?

This could be due to the following reasons:

• Pure dye component’s spectra are incorrect. Rerun pure dye spectra.
• Incorrect dye components have been chosen. Choose correct dyes for data analysis.

Which reverse transcription kit can I use with the PowerUp SYBR Green Master Mix (Cat. No. A25741, A25742, A25743, A25776, A25777, A25778, A25779, A25780, A25919)?

We recommend using the High-Capacity RNA-to-cDNA Kit (Cat. No. 4387406, 4388950).

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.

What primer concentrations can I use with the PowerUp SYBR Green Master Mix (Cat. No. A25741, A25742, A25743, A25776, A25777, A25778, A25779, A25780, A25919)?

The recommended primer concentration range is 300-800 nM. You will need to optimize the primer concentration by running different concentration combinations of the forward and reverse primer. See page 28 of the PowerUp SYBR Green Master Mix User Guide.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.

What type of samples is the MagMAX DNA Multi-Sample Ultra Kit (Cat. No. A25597) optimized for?

We have optimized protocols for extracting DNA from buccal swabs, whole blood, blood cards (Whatman FTA), oral rinse, saliva, and urine samples. In addition, the kit can be used to process vaginal microbiota samples (e.g., ThinPrep, Surepath) for gDNA extraction from yeast, bacteria (Gram positive/negative), viruses, and protozoa with the addition of additional enzymatic steps.

In the MagMAX DNA Multi-Sample Ultra Kit protocol, why do blood samples appear cloudy after adding the Multi-Sample DNA Lysis Buffer?

On rare occasions, the sample may turn cloudy after adding the Multi-Sample DNA Lysis Buffer. The sample will clear up when isopropanol is added. This will not have any impact on the yield or quality of the DNA.

Can heparin be used to collect blood samples for the MagMAX DNA Multi-Sample Ultra Kit?

Heparin is not recommended as an anti-coagulant since it can cause inhibition of PCR reactions. Instead, we strongly recommend collecting blood samples in EDTA or sodium citrate.

What is the best way to remove the buccal swabs from the MagMAX DNA Multi-Sample Ultra Kit lysate?

We recommend transferring the lysate to a new MagMAX Express-96 Deep Well Plate (Cat. No. 4388476). This option eliminates contamination risks and saves time. To transfer lysates, set a multi-channel micropipetor to ˜300 µL and transfer one row at a time. Each well should contain 200-250 µL after transfer.

Can buccal swabs that are to be used with the MagMAX DNA Multi-Sample Ultra Kit be shipped after sample collection?

Buccal swabs can be shipped at 25 degrees C or below. Exposure of the swabs to high temperatures (over 30 degrees C) during shipment has been associated with poor performance in the workflow. To prevent degradation, ensure that buccal swabs are completely dry before shipment. Place the swabs in a paper envelope for storage after collection - paper envelopes are permeable and allow the swabs to dry effectively.

What buccal swabs do you recommended using with the MagMAX DNA Multi-Sample Ultra Kit?

We recommend using one of the following types of buccal swabs with foam tips:

- Puritan PurFlock Ultra Flocked Swabs (Fisher Scientific, Cat. No. 22-025-192)
- Puritan HydraFlock Swabs, standard tip (Puritan, Cat. No. 25-3306-H )
- Sterile Foam Tipped Swabs (Puritan, Cat. No. 25-1506 1PF)
- 4N6FLOQSwabs, regular tip (Thermo Fisher Scientific, Cat. No. 4473979)

Note: Please do not use cotton swabs, as they may contain PCR inhibitors.

Why does the MagMAX DNA Multi-Sample Ultra Kit have two DNA elution buffers?

DNA Elution Buffer 1 is at a basic pH to help maximize release of the gDNA from the magnetic beads and has to be neutralized by DNA Elution Buffer 2.

Why is no RNase treatment included in the MagMAX DNA Multi-Sample Ultra Kit workflow?

RNase A treatment is not critical for this workflow. RNA that might be present in the samples is likely to be degraded in the course of the experiment and should not interfere with downstream applications. For those sample types that might potentially have some RNA carryover, we have included the RNase A as part of the Bead mix as a precaution.

Can I combine DNA Elution Buffer 1 and DNA Elution Buffer 2 from the MagMAX DNA Multi-Sample Ultra Kit and just have a single elution step?

Yes, you can combine the two elution buffers prior to eluting the DNA; instead of having the instrument pause, requiring you to add DNA Elution Buffer 2, press ‘start' on the instrument - just be sure to combine them directly before adding to the elution plate and before putting the plate on the KingFisher instrument (you do not want them to sit as a mix for too long). For example, if you wish to elute in 50 µL of buffer, you can mix 25 µL of DNA Elution Buffer 1 and 25 µL of DNA Elution Buffer 2, then add the total 50 µL into a well and run the protocol as you normally would.

Can I edit the manufactured KingFisher Flex protocols?

No, manufactured protocols are locked for editing, but they can be renamed using the ‘save as' function and edited as a new version.

Can I use magnetic beads from other vendors with the KingFisher Flex instrument?

For nucleic acid extraction, we recommend using our MagMAX kits and MagJET kits. Other magnetic beads should work well as long as they are 0.5 to 10 µm in size. However, you will have to write your own KingFisher protocols using the Thermo Scientific BindIt software and validate/optimize the protocol on your own.

Find additional tips, troubleshooting help, and resources within our Automated Nucleic Acid Purification Support Center.

I am working with liquid samples with high volume and want to extract nucleic acid from them using the KingFisher Flex instrument. Can I use more volume than the protocol advises?

Yes, this can be done by sample splitting. Set the protocol so that the magnet will collect samples from multiple wells before continuing to the next step. Be sure not to overload.

Find additional tips, troubleshooting help, and resources within our Automated Nucleic Acid Purification Support Center.

Can I scale up my reaction volume with the KingFisher Flex instrument?

Yes, reaction volumes can be scaled up, and all other volumes (i.e. wash volumes) will have to be increased by the same fold difference. It is generally not recommended to scale up reaction volumes more than 2-fold unless the plate's volume capacity will allow it.

Are all of the Applied Biosystems MagMAX kits validated for the KingFisher Flex Magnetic Particle Processor?

Yes, all MagMAX kits can be used with the KingFisher Flex Magnetic Particle Processor.