How to Melt a DNA Pellet After Extraction: Techniques and Best Practices

How to Properly Resuspend a DNA Pellet After Extraction

DNA pellets are not melted but resuspended to dissolve the DNA effectively. The term “melt” is misleading. Instead, resuspension involves carefully dissolving the dried pellet back into solution without degrading the DNA.

Terminology Clarification

After DNA precipitation and centrifugation, what remains is a DNA pellet. The goal is to resuspend this pellet, which means to disperse it in an appropriate buffer. Melting suggests heating to a phase change, which does not apply to DNA pellets.

Physical Agitation and Handling Techniques

• Add buffer such as TE (Tris-EDTA) or nuclease-free water directly to the pellet.
• Pipette up and down vigorously to break up the pellet.
• Gently poke the pellet using the pipette tip to facilitate dissolution.
• Flick the tube gently several times to promote mixing.
• Spin briefly in a microcentrifuge to collect liquid at the bottom before further manipulation.
• Place the tube on a slow rocker at room temperature overnight to enhance resuspension.

Temperature-Based Methods

Increasing temperature can improve resuspension efficiency:

• Incubate the tube at 56°C for 15 minutes to 1 hour.
• Leave the tube at 4°C or room temperature overnight to allow gradual dissolution.

Freeze-Thaw Cycles

Freeze-thawing can aid in resuspension by disrupting the pellet matrix. However, multiple cycles may impact DNA integrity, especially for applications requiring high molecular weight DNA, such as ultra-long read sequencing.

Alternative Wetting Strategy

Some practitioners have considered wetting dry DNA pellets with 70% ethanol before adding buffer. This strategy lacks widespread validation, but theoretically, ethanol may loosen pellet compaction before resuspension.

Verification of Resuspension

It is crucial to confirm resuspension success:

• Measure DNA concentration using spectrophotometry (e.g., Nanodrop).
• Run an agarose gel to check DNA integrity and confirm presence.

Summary of Best Practices

• Resuspend, do not melt, DNA pellets.
• Add TE buffer and agitate by pipetting or gentle flicking.
• Use temperature incubation (4°C overnight or 56°C for 15–60 minutes) to aid dissolution.
• Limit freeze-thaw cycles to preserve DNA quality.
• Verify resuspension by quantification and gel electrophoresis.

How should I resuspend a DNA pellet after extraction?

Resuspension, not melting, is the goal. Add TE buffer to the pellet. Then pipette up and down or gently flick the tube to help dissolve it.

Is applying heat necessary to resuspend the DNA pellet?

You can place the sample at about 56°C for an hour or leave it at 4°C overnight. Some prefer room temperature incubation overnight before heating briefly.

Do freeze-thaw cycles assist in resuspending DNA pellets?

Freeze-thaw cycles can help break up the pellet. However, avoid this if you need very high DNA integrity for methods like ultra-long read sequencing.

Can wetting a dry DNA pellet with ethanol help in resuspension?

Some try wetting the pellet with 70% ethanol before adding buffer. It might help but is less common and not widely confirmed.

How do I confirm the DNA pellet has fully resuspended?

Check the solution using a spectrophotometer or run a gel. This ensures the dissolved material is DNA and your resuspension worked.