#Sanger sequencing chromatogram viewer how to#

Another common problem is that when using a Nanodrop, if the range of A260 is not between 0.1 and 0.8, the reading will be inaccurate (sometimes by orders of magnitude). The template and primer DNA in the PCR tube will give you a spec reading regardless of whether you have a product in the tube. One is that quantifying a PCR sample before purification will lead to incorrect QA of your sample.

Quantification of concentration:Р’В We often see a few problems here that cane be easily overcome.

recommendation: post-PCR cleanup with a cleanup kit such as Qiaquick (Qiagen).

why:both PCR primers will work in your sequencing reaction and produce a mixed template sequence, buffers will impact performance of sequencing enzyme mix.

I s your PCR cleaned of all PCR reaction reagents (primers, enzyme, buffers etc)?.

recommendation: assess by gel electrophoresis for each set of PCR reactions that provide template submitted for sequencing.

why:multiple templates will give a mixed template sequencing reaction (if your negative has a band the same size as your target, you may have a mixed template in your target band).

Have you ensured your negative control PCR reaction (no DNA PCR) is blank?.

recommendation:assess by gel electrophoresis for each template submitted for sequencing.

why:multiple templates will give a mixed template sequencing reaction, low quality/quantity template may cause short sequencing reactions or failed sequencing.

Do you have a single product for your PCR?.

We have prepared a guide to help you assess your sequencing data and to identify potential causes of a failed reaction. For the vast majority of sequencing reactions, simply repeating the same reaction with the same template and primer mixture will not resolve a failed sequence. If these steps have been taken and a sequencing reaction still fails, there are specific ways to interpret your electropherogram and identify the cause of the failure. Luckily, there are a few simple steps one can take in the preparation of a sample for sequencing to minimize failed or low-quality sequencing results. Typically a robust technique, sequencing reactions can and do fail from time to time.

#Sanger sequencing chromatogram viewer how to#

Did you get a sequence electropherogram that does not look right?Р”Р„re you thinking of submitting a new sample and want to know how to prepare it?Р”Р„utomated Sanger sequencing remains the gold standard for single target DNA sequencing.