Sometimes a primer is not very efficient for sequencing.  Make sure you are sending the correct primer at the correct concentration (usually only 1-2 microMolar). 

You may need to send the other primer and sequence from the opposite end of your product. 

You could also redo the PCR using primers that flank your insertion and use those primers for sequencing.  They are usually very robust.

Hi Katie,

Thanks for your advice! I've used this primer for sequencing before (same gene, different vector), and it worked fine then. I'll try using the other primer, I hadn't thought of that. 

Do you mean primers that anneal to just the vector? I'm not sure if we have any, but I can ask around; that seems like it would work well. Thanks!

what method did you use to get rid of the pcr primers before sequencing the product please?

The following could be possible reasons: 1. Quality of your product (concentration of target gene). Sometimes, PCR may give you thin band (low quality or low concentration) but same will not work for sequencing. 2. Sometimes, if you have send your samples as well as primers specific to target genes to long distances, sufficient care should be taken that they may not degrade. Shipment in ice cubes is one of the crucial steps. 3. Bidirectional sequencing is highly preferable (i.e. sequencing with sense and antisense primers). 4. Methodology may be revisited.

Try to send your plasmids for sequencing with the primer outside of your gene (in the promoter region or standard sequencing primers depending on your backbone). If the sequencing does not work with these primers either, it should be the problem of the template, so I would re-do plasmid isolation and perform restriction digestion of your plasmids to see if the restriction pattern matches the product you expect to get. 

Probably these tips might aid you:

Primarily, PCR primers are designed for  PCR amplification to obtain an amplicon. While the Sequencing primers  to sequence a DNA fragment and reveal its DNA sequence identity. PCR being an exponential process, and  is usually carried well beyond completion, even poor primers produces amplification in a PCR reaction. Sequencing. however, is strictly linear, and is much more unforgiving of poor primers.

So I would design an additional primer for sequencing.

Cheers!

Try to use different PCR and sequencing primer. It works better always. When you sequence after PCR using same primer, there is always a risk for off target binding of the primer. 

• PCR primers should work fine for sequencing if they have amplified your product efficiently by PCR. I used to do this routinely all of the time
• Failure to sequence from your amplified PCR product might suggest issues relating to the quantity and purity of your amplicon
• How did your purify away salts dNTPs and primers before attempting sequencing ? I'm assuming you are removing impurities by column purification or SAP exo digestion ?
• If you know from running a small fraction on the gel that your product is just your one single amplicon then avoid gel extraction purification for sequencing - especially with the problems you describe - as bleed through of muco polysaccharides can inhibit amplitaq 
• My first piece of advice would be to run a small amount of your purified product (~10%) on an agarose gel to ascertain approximate amount and eliminate degradation problems 
• Then try using about 10ng/100 bp of purified product for Sanger sequencing