No Bands - Genotyping

Troubleshooting genotyping assays when you get no bands can be challenging.  The underlying problem can be any part of the PCR including the primers or other reagents, the DNA (quality and/or quantity), or the thermal cycling parameters.  Determining where the problem lies requires a methodical approach.  Following this approach will help you identify the problem, so that it can be fixed.

First, check your primers 

Solution

Rationale

Check sequence

Wrong sequence may not amplify

Check dilution

If primers are too dilute, they will not amplify

If the primer sequences and dilutions are confirmed to be correct, the next steps are to look at the DNA. If not, then you may need to re-order primers with the correct sequence, or optimize the primer concentration.  

Start by genotyping the DNAs for the strain giving you no bands with a genotyping assay for another strain that is currently working for you, along with the controls for that other strain that have worked previously. 

If you get no bands, then your problem is most likely with the DNAs. 

Solution

Rationale

Quantify DNA

Too much or too little DNA can lead to no bands. Use approximately 0.5 ng – 0.5 µg of total genomic DNA per 25 µl reaction.

Check 260/280 ratio of DNA

If the DNA quality is poor, it may not amplify bands

Try diluting DNA

To reduce contaminates that may interfere with amplification

Re-extract DNA using new reagents

 

If you do get bands, then the problem is not directly with the DNA.  The problem could be the thermal cycling parameters or the DNA extraction protocol. 

Solution

Rationale

Test an annealing temperature gradient

Find the optimal annealing temperature. If your annealing temp is too high, you may not amplify bands

Increase the number of cycles

Too few cycles can lead to no amplification

Use a PCR Additive

Reduce DNA secondary structure or non-specific priming

Try re-extracting DNA using a different protocol

Some primers may not work well with your normal DNA extraction protocol.Find DNA isolation protocols here

 


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

Make sure you are including controls that have previously worked.  If not, re-genotype with such controls.

If the previous controls work as expected but new unknown samples give no bands, the problem is likely with the new DNAs.  Follow the same solutions as the previous section for resolving DNA quality/quantity issues.

If you get no bands for any DNAs, you likely have a problem with your primers. 

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.