Thermolabile UDG

What it means: Inactivation Temp is the temperature above which the enzyme is destroyed. Inactivated above 50°C means this glycosylase is rapidly and completely killed once the reaction is warmed past 50°C, and stays active below it.

Why it matters:

• A carryover-prevention step only works if the UDG is gone before the new amplicon accumulates — otherwise it destroys the product it was meant to protect.
• Standard, heat-stable UDG retains residual activity through cycling and can degrade dU-containing new product; a thermolabile enzyme cannot.
• Inactivation happens during the normal initial denaturation, so no extra step is added to the run.

How to interpret here: Run the UDG treatment at room temperature before amplification, then move straight into cycling — the initial denaturation destroys the enzyme. No separate inactivation hold is needed.

Tip: Do not warm the reaction above 50°C before the UDG treatment has had time to act, or the carryover step is skipped entirely.

What it means: Substrate describes what the enzyme acts on. Here it is dUTP-containing amplicons — PCR products made with dUTP substituted for dTTP, so that uracil marks them as previous-round material.

Why it matters:

• The enzyme only recognises uracil, so genuine dT-containing sample DNA is untouched while contaminating dU amplicons are destroyed.
• This selectivity is the whole basis of carryover prevention: the contamination is chemically labelled, not just diluted out.
• It requires the whole workflow to use dUTP — a reaction run with normal dTTP produces product this enzyme cannot recognise later.

How to interpret here: Substituting dUTP for dTTP must be adopted across the assay from the start. Removing uracil creates abasic sites that block extension, so contaminating amplicons cannot be re-amplified.

Tip: Uracil-containing product also cannot be used for downstream cloning into some systems — plan the workflow before switching.

What it means: Buffer Included describes the reaction buffer supplied with the enzyme. This product ships with 500 µl of 10X UDG Reaction Buffer alongside 200 reactions (100 µl) of enzyme.

Why it matters:

• The buffer is formulated for this glycosylase, so pH and salt are already at the enzyme's optimum for the room-temperature treatment step.
• Supplying it in the pack means the carryover step can be run without sourcing a separate buffer.
• Buffer volume is scaled to the enzyme pack, so nothing runs out mid-project.

How to interpret here: Dilute the 10X buffer ten-fold in the treatment reaction. The UDG step is normally performed before the PCR master mix is completed, not inside it.

Tip: Thaw and mix the buffer completely before pipetting; components settle during frozen storage.