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.