MFEprimer-4.2.4: PCR Primer Design and Quality Control Tools

Notice: Long amplicons can cause very long design times and server instability. Your account limits: target lines ≤ 100, amplicon length ≤ 5000 bp. Default public cap is 5000 bp; paid accounts may be raised on request. Feedback: quwubin@gmail.com.
重要通知：长扩增子可能导致设计耗时过长、服务器不稳定。当前账号限制：靶区行数 ≤ 100，扩增子长度 ≤ 5000 bp。公共默认上限为 5000 bp；付费账号可申请提高。反馈：quwubin@gmail.com。

Introduction Parameters help Examples

Target settings

Genome database

Targets in BED format (<= 100?, example, clear, SNP, Gene how-to)

STR mode: primers should be chosen around (not within) the regions.

Chromosome name examples

chrM    1000 1001
chrM    2000 2001
chrM    3000 3001

Validation method NGS or Gel electrophoresis

Product Size Min Difference

Only for size discrimination method, e.g., gel electrophoresis.

Quality control settings 1

Non-specific Amplicon T_m

Min

°C

The minimum binding stability between primer and its binding sites.

Non-specific Amplicon Size (bp)

From

Product size: 0 (min) - 1000000 (max).

Number of Off-targets

Min

Max

Number of non-specific amplicons.

Dimer ΔG

Min

kcal/mol

Primers with dimer stability exceeding this threshold will be discarded.

Hairpin Score (base pairs)

Min

Primers with hairpin stability exceeding this threshold will be discarded.

Minimum Nucleotide Differences

Min

Minimum number of different bases between target amplicon and off-targets if allowed.

SNP Skip Size

Min

SNP-free region length from primer 3' end.

InDel Skip Size

Min

InDel-free region length from primer 3' end

Quality control settings 2 (optional)

Background Genome 1

Background Genome 2

Amplicon Count on Background Genomes

Min

Max

Primer settings

Primer Min Size

Primer Opt Size

Primer Max Size

Primer Min GC

Primer Opt GC

Primer Max GC

Primer Min T_m

°C

Primer Opt T_m

°C

Primer Max T_m

°C

Product settings

Product Min GC

Product Opt GC

Product Max GC

Product Min Size

扩增子长度不能超过 5000 bp / maximum 5000 bp

Product Max Size

Account amplicon length limit: 5000 bp. 账号扩增子长度上限：5000 bp。

扩增子长度不能超过 5000 bp / maximum 5000 bp

Thermodynamic settings

Concentration of monovalent cations (usually KCl)

Concentration of divalent cations (usually MgCl₂)

Concentration of dNTPs

Annealing oligo concentration

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