One Band Not Amplifying

Having one of the expected bands in a genotyping assay fail to amplify well or at all can be frustrating. If that band is the mutant or transgene band, it is easy to assume that the mice are wild type and not carrying the mutation or transgene as expected. However, you will want to work through potential solutions to make sure you are not jumping to the wrong conclusion.

If this is a new assay to you….

Start by checking your order and your primer(s) specific for the band that does not amplify.

Solution	Rationale
Review your order confirmation	Make sure you received the correct strain and genotype that you expected
Check primer sequence	Wrong sequence may not amplify
Check primer dilution	If primers are too dilute, they will not amplify

If your order and primers check out, then re-genotype using two separate reactions; one for the mutant allele or transgene, and one for the wild type allele or internal positive control.

If both bands amplify when separated but not when multiplexed, try altering the primer concentration for the primer or primers specific to the band that does not amplify when multiplexed.

Solution	Rationale
Increase amount of primers specific for band that does not amplify	When multiplexing, one band can be preferentially amplified.
Decrease amount of primers specific for band that does amplify	Same as above
Both above at same time	Same as above

If the band fails to amplify even when separated, the problem could be the thermal cycling parameters or the DNA extraction protocol.

Solution	Rationale
Test an annealing temperature gradient	Find the optimal anneal temperature. If your annealing temp is too high, you may not amplify bands
Increase the number of cycles	Reduce DNA secondary structure or non-specific priming
Use a PCR Additive	Too few cycles can lead to no amplification
Try re-extracting DNA using a different protocol	Some primers may not work well with your normal DNA extraction protocol.

If the assay previously worked, and now does not….

Look to your specific primers or a potential breeding error with the parents of the mice that are not genotyping as expected.

Solution	Rationale
Make a new working dilution from your concentrated stock	Primers can degrade when maintained at lower working concentrations or following repeat freeze/thaws.
Re-order primers	Primers can degrade when maintained at lower working concentrations or following repeat freeze/thaws.
Re-extract DNA from the parents and re-genotype	If the parents are not the expected genotype, then the mice may not be the expected genotype. The assay may actually be working correctly.