As the quality of sequencing results depends greatly on the purity and integrity of the template DNA, it is very important that careful attention be given to the preparation and quantification of the DNA to be sequenced. Generally, the use of the commercially available DNA purification kits to prepare the templates will yield sequence quality DNA. Please contact us if you have questions about DNA purification, DNA preparation, submission volumes or sample placement in a 96-well plate.

Universal primers can be added by the Division staff if the primers are requested on the submission form (CORE.T7, CORE.T3, etc.). If a core (CORE.*) primer is not requested on the submission form it will be assumed that you have added your own primer.

Individual Tube Submission Protocol

The template and primer requirements are as follows:

  1. Amount of template DNA per reaction should be 0.5 ug for ds DNA or 0.5 ug ssDNA (template = plasmid or PCR product that is 1.5 to 8 kbp).
  2. For PCR fragments less than 1.5 kbp, use 10 ng/100 bp of amplified product.
  3. For templates larger than 8 kbp, submit 1 ug.
  4. Submit 6-10 pmoles of one primer* per tube if your template is greater than 1.5 kbp. Submit 10-15 pmoles of primer if your template is less than 1.5 kbp.
  5. Please bring the total volume up to the amounts listed below with ddH2O.
  6. Sample should be provided in a 0.5 ml microcentrifuge tube.
  7. Label top of 0.5ml tube with 5-digit order number and sample number.  Samples should organized in numerical order. The DNA sample should be submitted as follows:
  • Single Tube submission with your custom primer:  Template + Your Primer = 11.0 ul total volume
  • Single Tube Submission, IIHG Genomics Division adds primer: Template in 7.0 ul (core will add indicated Core Primer in 4.0 ul to give the 11.0 ul total volume)

Plate Submission Protocol

The template and primer requirements are as follows (note volumes are less for plate submissions):​

  1. Amount of template DNA per reaction should be 0.25 ug for ds DNA or 0.25 ug ssDNA (template = plasmid or PCR product that is 1.5 to 8 kbp).
  2. For PCR fragments less than 1.5 kbp, use 5 ng/100 bp of amplified product.
  3. For templates larger than 8 kbp, submit 0.5 ug.
  4. Submit 3-5 pmoles of one primer* per well if your template is greater than 1.5 kbp. Submit 5-7.5 pmol of primer if your template is less than 1.5 kbp.
  5. Please bring the total volume up to the amounts listed below with ddH2O.
  6. Sample should be provided in a 0.2 ml ½ skirted plate. (ALL plates must be GeneAmp 9700-compatible) 
  7. Seal plate using plastic adhesive film.
  8. Please label plate with 5 digit order number. The DNA sample should be submitted as follows:
  • Plate submission with your custom primer: Template + Your Primer = 5.5 ul total volume
  • Plate submission using our core primers is not available. You must provide your own primers for plate submissions.

 

​We are able to run full or half-full 96 well plates. If using only half a plate, please load samples into odd numbered wells. Please download and complete the appropriate Plate Record for either full or half 96 well plates. Sample and primer names should be entered in the appropriate well position on the plate record. Blank/empty wells should be filled in with the labels “Empty1”, “Empty2”, etc. All sample primer name combinations must be unique. You may only letters, numbers, and periods (.) in your sample and primer names. Please note that the following characters cannot be used in sample/primer names:

@ # % ^ & * (  ) + =  \ / : ; " < > | ? ' , - _ SPACE

The 96 well plate format is recommended for researchers who are confident in their methodology for template and primer preparation as all samples submitted on the plate, regardless of the results, will be charged. Plate submissions will be charged in multiples of 48 reactions. 

All high volume plate submissions must be in half-skirted 0.2 ml PCR plates. Plates can be acquired from Biochemisty Stores (4-321 BSB), Biomedical Research Store (238 EMRB), or  they can be ordered elsewhere. Please contact us if you have questions regarding plate selection.

57655 96-Well Optical Reaction Plates (20ct) # 4306737 1/box ABI-Life Tech – available at the Biomedical Research Store (238 EMRB)

1007473 Plates PCR 96-Well THERMOWELL Half-Skirt PP  CS/25 PKG Tech – available at the Biochemistry Stores (4-321 BSB)

1007468 Plates THERMOWELL Skirted Universal PP {USA} CS/10 Tech – available at the Biochemistry Stores (4-321 BSB)

Sample Submission Tips for Success

Prepare samples by adding the appropriate amount of template and primers to water (i.e., not TE or Tris buffers). We highly recommend the purified template to be suspended in water or a diluted TE (2 mM Tris + 0.1 mM EDTA). Water or diluted TE should be used to elute templates from the purification columns or suspend templates following ethanol precipitation.

Sequencing results have been found to be inconsistent when template and/or primer are dissolved in TE (10 mM Tris and 1.0 mM EDTA). EDTA chelates Mg2+ ions and subsequently inhibits our reaction enzyme. Reducing the EDTA to 0.1 mM will not inhibit the enzyme and will give more consistent sequencing results.

Also, the use of buffers such as 10 mM Tris can inhibit sample loading into the sequencing machine due to the electrophoretic loading by the machine. We suggest using 2 mM Tris as a buffer for template and primers stocks. We highly recommend using water to elute template and primers for sequencing.

We add the following to the sequencing reaction before cycling: 0.5 ul DMSO, 0.45 ul of Applied Biosystems Sequencing Big Dye Terminator (BDT) Mix, 0.05 ul dGTP BDT reaction mix, 2.0 ul 2.5x buffer, 1.5ul water, 5.5 ul Template and primer to give a 10.0 ul Total reaction volume.

The division utilizes cycle sequencing. This is not the same as PCR. Cycle sequencing is a linear amplification of DNA, hence the need for only a SINGLE primer. Addition of TWO primers will result in data with multiple signals that is NOT interpretable.


Contact Information

116 EMRB
Phone: (319) 335-6736
Fax: (319) 335-6737
dna-sequencing@uiowa.edu

Samples for sequencing may be dropped off at 116 EMRB anytime from 8:00 AM to 5:00 PM Monday-Friday.