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Laboratory of Biotechnology and Bioanalysis Washington State University

FAQs

How do I get Big Dye reaction mix to do my own sequencing reactions? 

Please visit our lab to get some mix for your lab. We don’t mind providing you a large aliquot if you or your lab always has sequencing to do. You will need 4ul/reaction. The mix is quite stable when stored at -4c. Always protect the mix and the sequencing reaction products you make from light. The highly fluorescent dideoxy terminators, as you might expect, are quite photo sensitive. 

We have shipped big dye mix to the branch campuses before without cooling via interdepartmental mail. This process takes a couple of days, but it will still work since the DNA polymerase in the reaction has highly thermal stability. If you are not on the Pullman campus, investing in your own mix is an easy solution. We will only charge you for the data collection run. 

 

How do I perform the Sanger sequencing reaction? 

Even though we are using a thermal-cycler, the reaction it is not a PCR. It is critical that only one primer and one priming site are present in your reaction. If you are sequencing a PCR product, you must get rid of excess primers and nucleotides by using a PCR cleanup column, gel filtration, SPRI beads, precipitaion, or by enzymatic degradation using ExoI and SAP. Exo-SAP-IT is the easiest method by simply adding a ul containing a sexonuclease I and a heat labile shrimp alakaline phophatase. Next, incubate at 37C for 15 minutes, and finally heat kill the enzymes at 80C for 15 minutes before using in a labeled dieoxy terminator reaction. The  new Exo-SAP-IT “express” formulation only requires 4 minutes at 37C and 1 minute at 80C. 

 

How to build the reaction:

DNA Template concentration matters! Know your template quality/quantity. 

Combine: 

  • 200-500ng if sequencing from a dsDNA replication vector DNA or 40-90ng if you are sequencing from ssDNA vector or PCR product 
  • 3.2pmol of one oligonucleotide primer
  • 4ul of big dye terminator sequencing mix water to a final volume of 10ul

Thermal-cycling: 

Sanger reaction cycling is not a normal PCR. It needs a long low extension for best incorporation: 

  • 96C 10 seconds
  • 50C 15 seconds
  • 60C 4 minutes 

Repeat 25 cycles 

 

Reaction Clean-up: 

Unincorporated labeled nucleotides in the reaction must be removed before sequence analysis by using a PCR clean-up column, size exclusion, SPRI beads, or by ethanol precipitation of the products, and the final sample is then dried in a speed vac. If you do not have a speed vac we can do this for you. 

Our Sanger sequencing service ranges from performing the whole process starting from your primer and template, to only running the final products. Many of our users cycle and cleanup their own samples. We can do as much or as little of the process as you need. 

 

How do I get my samples to you? 

Walk-in: 

The Biotechnology/Life Sciences Building is right across Stadium Way from the bronze cougar statue. Entrances are on the east side where the surrounding buildings connect. The yellow parking lot next to the building has a day permit kiosk near the baseball field entrance. 

There is a small sample refrigerator when you walk-in to the lab. Place well-labeled samples into the refrigerator and fill out a sample sheet next to the refrigerator or email this to LBB. 

Mail:   

DNA is pretty stable at room temperature buffered with a little EDTA. Standard DNA re-suspension buffer is TE (10mM Tris pH8 and 1mM EDTA). RNA should be shipped frozen.   

WSU/LBB 
Central Receiving and Delivery 
100 Dairy Road 
PO Box 7520 
Pullman, WA 99164-7520