DNA Sequencing

What happens in DNA sequencing?

The videos linked below are a good introduction to DNA sequencing.

DNA Sequencing - 3D

Next Generation Sequencing - An Introduction

DNA sequencing machines are very expensive and in our research lab we send our samples to EuroFins Genomatics for sequencing. This company has a 24 hour return date after the sample is recieved and will send the sequence information digitally.

Materials


Purifying PCR Product (via ExoSAP-IT Protocol)

  1. 1. Remove ExoSAP-ITTM reagent from –20°C freezer and keep on ice throughout this procedure. Keeping this reagent on ice is very important, it degrates quickly!

  1. 2. Mix 10 μL of a post-PCR reaction product with 4 μL of ExoSAP- ITTM reagent for a combined 14 μL reaction volume. When treating PCR product volumes greater than 10 μL, simply increase the amount of ExoSAP-ITTM reagent proportionally.  

  1. 3. Incubate at 37°C for 15 minutes to degrade remaining primers and nucleotides.  

  1. 4. Incubate at 80°C for 15 minutes to inactivate ExoSAP-ITTM reagent.  

  1. 5. The PCR product is now ready for use in DNA sequencing, SNP analyses, or other primer-extension applications. Treated PCR products may be stored at –20°C until required. 


Sequencing PCR Products

  1. 6. In an epitube, mix 10uL of purified PCR product and either your forward or reverse primer.  

  1. 7. Label the tube with your initials and some way to identify what the sample is.  

  1. 8. Store the tubes in the freezer and/or send them to EuroFin Genomatics for sequencing. 


EuroFin will send tubes and a spreadsheet so you can identify your samples.