AWRI Thum

Tips for successful DNA sequencing

General: The sequencing and genotyping facility can operate like a core facility that you submit your template to and the staff can perform all the necessary molecular work for you. However, it is also designed to be user-operated by faculty and students at GVSU. The more bench work you put into your samples, the more control you have over them and the cheaper it is to sequence them. AWRI's facilities are open for use or, if you choose, most of the required work can be done at your own bench!

 

Template: I see more successful sequencing reactions from PCR products than I do from plasmids. If at all possible, I'm always going to recommend using PCR products over plasmids. It might not always the best thing to do, financially as well as molecularly, especially when trying to sequence through the poly-A regions found in cDNA libraries (taq slips and increases background noise). Templates are best when suspended in water or buffers free of EDTA. If you are having difficulty getting the template to the required concentrations, please talk to Dustin. There are a few do's and don'ts and rules that we can bend to get you what you need.

 

Check PCR success: I would recommend that you check every PCR product on a gel to ensure you are amplifying a single locus and know the expected size before you clean-up the reaction. Sequencing chemistries are rather sensitive to contaminating products which, at the very least, increase background noise on the chromatograms.  Knowing the size of your fragment is also critical for optimizing the amount of PCR product that goes into the sequencing reaction. For routine sequencing of robust primers and clean template, it may not be necessary to run a gel of every sample.

 

Purify the PCR products: For optimal results, it is important to purify PCR products before sequencing. This can be done in a variety of ways; anything that removes dNTPs and primers will work. Two common ways are spin columns or enzyme digestion. ABI recommends using Centricon-100 columns but there are many other competitors to choose from. Enzymes are a cheap alternative to kits to clean up PCR reactions: Exonuclease I, which destroys single stranded DNA (ie unincorporated primers) and SAP (shrimp alkaline phosphatase or some variation) which renders the unincorporated dNTPs useless. Spin columns would be my recommendation, so that the samples can be spec'd to accurately quantify the DNA.

 

Quantify the Template: The grant that purchased the genetic analyzer also purchased a NanoDrop 1000 which is capable of spec'ing as little as 1ul of undiluted sample. This allows you to quantify your samples so you know how much to add to the BigDye sequencing reaction. Keep in mind that an enzyme clean-up of a PCR reaction will not give you an interpretable reading as many things that absorb at 260nm are still with the samples. Alternatively, you can quantify PCR products on an agarose gel using appropriate standards.

 

Sequencing reactions: The sequencing facility routinely does 1/8 BigDye v3.1 sequencing reactions without problems (Total volume is 5ul). The most important part of the sequencing reactions is going to be the purity/concentration of the template. Sequencing reactions are essentially a PCR, and are carried out inside a thermalcycler.

 

Clean-up Sequencing Reactions: As with everything, there are options. We are set-up to do sephadex clean-ups at AWRI and the reagents/materials to do so are included in the cost to run a sample on the sequencer. We do these in plate form, so up to 192 samples (2 plates) at a time. There are alternatives to use but I'd recommend sticking with sephadex and which you or the sequencing facility's staff can perform. If you’d like to do your own clean-ups, please contact Dustin before submitting samples.

 

Run the Instrument: The only thing left to do is actually put the samples on the instrument and collect the data! A technician will always be available to double check the instrument and make sure nothing will break during the run.

Page last modified March 11, 2014