School of Freshwater Sciences
Great Lakes Genomics Center
School of Freshwater Sciences

Call: (414) 382-1774

3730 Sanger Sequencer

3730 Sanger Sequencer

Our Sanger sequencing services can process plasmid, PCR products, cosmids, phage, and BAC DNA samples. Our 3730 routinely provides up to 800 bases of usable sequence per reaction. We also provide genotyping services for researchers conducting studies with fluorescently labeled primers. We can process microsatellites, AFLPs, and SNPs. All samples must be submitted in either a single tube or 96 well plate format. We offer 2 levels of service: full service and ready to load plates.

Full Service Sequencing: User supplies template and primer pre-mixed according to tables below. We will set up, cycle, clean and analyze the reaction. Submit > 24 samples in a 96 well plate. Number each tube or well and if sample names other than the labeled number are needed, provide a corresponding MS Excel spreadsheet. If more than 1 primer is being used for 1 template, a separate template/primer mix needs to be set up for each primer. Each unique template/primer mix is considered to be 1 reaction. BigDye v3.1 is used for all sequencing reactions.

Template
Plasmid Prep 300-600 ng/reaction (final concentration 100-200 ng/ul)
PCR fragment 10-20 ng/reaction (final concentration 3-8 ng/ul)
Primer 10-20 pmol
(for all types of reactions)
Mix Together for 1 reaction:
Template 1-6 μl
Primer 0.8 μl

Ready to Load Plates – Sequencing and Fragment Analysis: Client performs their own reactions in a 96–well, half-skirted plate, cleans up the reaction, and re-suspends samples in at least 10 μl of formamide. Wells that do not contain a sample to be sequenced must contain 10 μl of nuclease free water. Please be sure to use plates compatible with an ABI 3730 DNA Analyzer. We recommend these: Dot Scientific, catalog # 951-PCR. Plates should be delivered to the Genomics Center foil sealed and well labeled. If a sample name other than the well position is needed, provide a corresponding MS Excel spreadsheet organized by columns, ie A1,B1,C1, etc. The plates can only be loaded onto the sequencer in one direction and well position is automatically assigned to the sequence. It is very important to have your MS Excel spreadsheet laid out correctly if you need a sample name assigned to each well position. Contact the Genomics Center with questions or to verify your sequencing plan. Training is available from GLGC if you would like to set-up your own sequencing reactions and prepare a ready to load plate.

Please contact GLGC 1 day in advance so that we can be ready to load your plates onto the sequencer when they are delivered to the lab.

Things to keep in mind:

  • The most important factor in DNA sequencing is staring with a clean, pure template. We do not recommend, and re-sequencing policies will not apply when, sequencing a template with a 260/280 ratio below 1.8 and 260/230 ratio below 1.6.
  • Dilute template in water not TE buffer.
  • PCR primers need to be removed before the Big Dye Terminator sequencing reaction can be set up. We recommend Qiagen’s QiaQuick PCR clean-up kit. Please also make sure that you only have 1 product from the PCR reaction.
  • Good PCR primers do not always make good sequencing primers. When sequencing PCR products, you may want to consider 2 different primers for sequencing the region of interest or sequencing from both directions.
  • You can expect 500-800 bp of high quality sequence when staring with high quality template. However, the first 25 – 50 bases are never high quality sequence so primers need to be designed accordingly to be sure you have good sequence over your region of interest.
  • It is difficult to sequence PCR fragments that are less that 100bp due to limitations of the BigDye v3.1 Terminator chemistry and many PCR column – based clean up techniques.

If you need to sequence a small PCR fragment please contact Angie Schmoldt.

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