Fungal DNA Barcoding Primers are short specific single-stranded DNA fragments (oligonucleotides) that define the region amplified during PCR during fungal DNA barcoding workflows.

The primers amplify:

• ITS1F/ITS4: The Internal Transcribed Spacer (ITS) region for Fungi.

The primers come in two formats: 

• A beginner-friendly single-tube Primer Mix (400 µL) that contains forward and reverse primers for simple and convenient PCR setup
• A Primer Pair containing forward and reverse primers in separate tubes (2 x 200 µL) for PCR and Sanger sequencing

Primer Mixes

• A single-tube format: Fewer tubes to track, and less pipetting.
• Ready-To-Use: No need to rehydrate your primers — just add to your PCR mix (containing your master mix, water, and DNA template).
• Convenient: Save on time and pipette tips with a premixed solution.
• Easy: No need for dilution calculations with a 10X concentration —  add 2 µL to a 20 µL reaction

Primer Pairs:

• A two-tube format: Individual forward and reverse primer tubes.
• Versatile: Use pairs of primers for PCR, and individual primers for forward and reverse Cycle Sequencing/Sanger Sequencing.
• Ready-To-Use: No need to rehydrate your primers — just add to your PCR mix (containing your master mix, water, and DNA template).
• Standard working concentration: Supplied at a concentration of 10 µM.

For use with DNA Barcoding PCR Protocol (Primer Mix), or DNA Barcoding PCR Protocol (Primer Pair).

Use these primers to amplify the standard DNA barcoding region for fungi: the Internal Transcribed Spacer (ITS) region.

Individual primers in the Primer Pair set can also be used as forward or reverse primers for Sanger Sequencing.

Primer mixes are formulated as an 8 µM primer mix in low TE buffer (10 mM Tris-HCl pH 8, 0.1 mM EDTA), suitable for single-tube setups.

Primer pairs are formulated as 10 µM individual primer tubes in low TE buffer (10 mM Tris-HCl pH 8, 0.1 mM EDTA) suitable for batch PCR setups. Individual primers can also be used for forward or reverse sequencing by Sanger sequencing services.

Recommended Usage

Primer mixes: 

Add 2 μL of primer mix per 20 µL PCR reaction to achieve a 0.8 µM final concentration. Add DNA template and PCR master mix as per the PCR protocol, and top the reaction up with PCR-grade water to a final volume of 20 µL.

Note: as little as 0.5 µM primer mix/rxn can be used if desired (to achieve a 0.2 µM final concentration), but this requires the use of a 2.5 uL pipette or a batch PCR mix approach as described for the primer pairs below.

Primer pairs: 

Primers are typically used at between 0.1 µM to 1 µM final concentration. We recommend 0.2 µM for typical usage.

In a 20 µL PCR, use 0.4 µL of each primer per PCR to achieve a 0.2 µM final concentration.

Due to the small volumes of primers used per rxn, we suggest making batch PCR mixes (e.g. 5, 10, 20, 32 rxns) and aliquoting 20 µL of the complete PCR mix into PCR tubes before adding DNA template to each PCR, as shown in the table below.

An additional 10% volume of all components has been added to the PCR mix table below to compensate for any pipetting errors during aliquoting.

If you wish to alter the volume of primer used, or to use any PCR additives, add or subtract the additional volume from the volume of PCR-grade water as appropriate.

Components	1 rxn (µL)	5 rxn* (µL)	10 rxn* (µL)	20* (µL)	32* (µL)
PCR grade water	13.2	56	145	290.4	464.64
5x Mastermix	4	22	44	88	140.8
Forward primer	0.4	2.2	4.4	8.8	14.1
Reverse primer	0.4	2.2	4.4	8.8	14.1
DNA Template (total)	2	10	20	40	70.4
Final volume	20	100	200	400	640
Excess	0	10	20	40	70.4

Primer sequences

ITS1F/ITS4

Forward: ITS1F: 5’-CTTGGTCATTTAGAGGAAGTAA-3’

Reverse: ITS4: 5’-TCCTCCGCTTATTGATATGC-3’

Expected amplicon length

500-1000 bp, depending on species.

Components

Primer Mixes: 10x stock solution: Mixed forward and reverse primers at 8 µM concentration, in low TE buffer (10 mM Tris-HCl pH 8, 0.1 mM EDTA)

Primer Pairs: Individual forward and reverse primers at 10 µM concentration, in low TE buffer (10 mM Tris-HCl pH 8, 0.1 mM EDTA).

Storage & Stability

Unopened primers should be stable at room temperature for several months. Opened primers can be refrigerated at 4 °C between uses, but should be frozen at -20 °C to extend shelf life.

Shipping conditions

Shipped at room temperature.