Long Non-Specific PCR Products

Troubleshooting PCRs - Long Non-Specific Products

This is a troubleshooting page for long non-specific PCR band problem and its solution.

Long Non-Specific Products or Bands Problem

Long Non-Specific PCR Products are usually encountered when you run your PCR reaction on an agarose gel and you spot larger non-specific PCR bands that are not the right size (or there are some or many bands larger than your target band size).

Long Non-Specific Products or Bands Solution

• Use less primer
• Decrease the annealing time
• Decrease the extension time
• Decrease the extension temperature to 62-68ºC
• Increase the KCl (buffer) concentration to 1.2x-2x, but keep MgCl2 concentration at 1.5-2mM
• Increase the MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant.
• Use less DNA template
• Use less Taq polymerase
• If none of the above works: check the primer for repetitive sequences (BLAST align the sequence with the databases) and change the primer(s)
• Use a combination of some/all of the above

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