DNA Core Facility

Services

• BioAnalyzer

  BioAnalyzer services include running DNA samples on an Agilent High Sensitivity DNA chip and RNA samples on an Agilent RNA 6000 Pico chip.

  DNA samples submitted to the DNA Core need to be diluted to 0.5ng/uL, while RNA samples need to be diluted to 5.0ng/uL. Samples containing a 5 uL aliquot a should be submitted in separate tubes numbered consecutively starting with number 1 on the top of the lid of your first sample tube with indelible ink. Both DNA and RNA chips have up to 11 sample wells that can be used. All samples submitted must be accompanied by a completed Sample Submission Form.

  The cost of the High Sensitivity DNA chip run is $80. The cost of the RNA 6000 Pico chip run is $48. Contact us for additional information.

• DNA and RNA Quantification

  We offer DNA and RNA sample quantification using the Invitrogen Qubit 2.0 Fluorometer. As little as 1-2 microliters is used for quantification. All samples submitted must be accompanied by a completed Sample Submission Form. The cost is $2.00 per sample.

• PCR Product Cleanup

  We offer ExoSAP-IT™ Express PCR product cleanup. A 5 uL aliquot of PCR product should be submitted in a 200 uL PCR tube. All samples submitted must be accompanied by a completed Sample Submission Form. The cost is $2.00 per sample.

• Level 1 DNA Sequencing on Applied Biosystems 3130XL Genetic Analyzer

  We have three 3130XL Genetic Analyzers set up for Sanger sequencing and fragment analysis. Different levels of service are provided, depending on your needs.

  Level 1: $4.00 per sample; <24 hour turnaround

  This is a load-only service for clients who perform their own cycle sequencing reactions using Life Technologies BigDye Terminator V3.1 Cycle Sequencing kits.

  Samples must be submitted in 0.2 ml PCR tubes. If possible, they should be submitted in completed batches of sixteen. All samples submitted must be accompanied by a completed Sample Submission Form.

  The amount of template DNA recommended to perform your own cycle sequencing reactions will depend on the quality of your product and the chemistry used. Highly efficient chemistries (such as BigDye V3.1) require less template. In general, we recommend using the following DNA concentrations shown below for best results depending on your template.

  Template	Quantity
  PCR product: 100-1000 bp >1000 bp >2000 bp	50-75 ng 75-100 ng 100+ ng
  Double-stranded (Plasmid DNA)	250-500 ng
  Cosmid, BAC	0.5-1.0 μg
  Bacterial genomic DNA	2-3 μg

  The facility recommended cycle sequencing reaction is shown below.

  Big Dye Terminator V 3.1
  Template	1.0 ul
  5X Buffer	2.0 ul
  Big Dye Mix	1.0 ul
  Primer (1.87uM)	0.5 ul
  Water	5.5 ul

  The facility recommended standardized cycle sequencing conditions are shown below.

  Cycle Sequencing Conditions on MJ Research Thermal Cycler
  15 seconds @ 95°C	Step 1
  10 seconds @ 50°C	Step 2
  4 minutes @ 60°C	Step 3
  	Go to Step 1, 35X
  4°C	End

  Samples for submission must be numbered consecutively starting with number 1 on the top of the lid of your first sample tube with indelible ink.

  Sequencing sample results are sent to the client within 24 hours using the email provided on the submission form. Sample files will have the standard (.ab1) extension.

  If a sample fails to produce any readable sequence due to a technical error on our part, it will be rerun automatically, using the original sample at no additional charge.

• Level 2 DNA Sequencing on Applied Biosystems 3130XL Genetic Analyzer

  We have three 3130XL Genetic Analyzers set up for Sanger sequencing and fragment analysis. Different levels of service are provided, depending on your needs.

  Level 2: $9.00 per sample; 48 hour turnaround time

  This is a service for clients that perform their own PCR or Plasmid Maxiprep/Miniprep but request that the facility perform the cycle sequencing reactions.

  Samples must be submitted in 0.2 ml PCR tubes. If possible, they should be submitted in completed batches of 16. All samples submitted must be accompanied by a completed Sample Submission Form and Level 2 Sequence Submission Form.

  Clients must also submit their sequencing primers in separate 0.2 ml PCR tubes each at a concentration of 1.87uM. We have had much success with primers ordered from Integrated DNA Technologies.

  PCR Samples submitted must have already have gone through a cleanup procedure and need to be accurately quantified in ng/μL. Clients who request cleanup of PCR samples will be charged an additional $2.00 per sample for the ExoSAP-IT PCR cleanup.

  After the cleanup procedure, samples must be accurately quantified. Clients who request DNA quantification of their cleaned-up PCR product will be charged an additional $2.00 per sample.

  Sequencing sample results are sent to the client within 48 hours using the email provided on the submission form. Sample files will have the standard (.ab1) extension.

  If a sample fails to produce any readable sequence due to a technical error on our part, it will be rerun automatically using the original sample at no additional charge.

• Fragment Analysis

  $4.00 per sample (<24 hour turnaround time)

  This is a load-only service for clients who perform their own genetic marker analysis experiments which include Microsatellites, RFLP, AFLP, etc. We use the ThermoFisher DS-30 Dye Set which includes 6-FAM, HEX, NED, & ROX.

  Samples must be submitted in 0.2 ml PCR tubes. If possible, they should be submitted in completed batches of 16. All samples submitted must be accompanied by a completed Sample Submission Form.

  Clients must purchase their own DNA size standard which is added to their sample before submitting them to our facility. We have had great success with the MapMarker size standards by BioVentures Inc., which are compatible with our POP-7 and 36cm capillary array setup. ThermoFisher's Hi-Di Formamide will be provided free of charge in 500 uL aliquots to ensure the highest quality results. The following reagents should be mixed as listed below.

  The amount of template DNA recommended must be determined empirically by the client. This can be achieved using a 1:10 dilution of the DNA sample and then a 1:50 dilution of the same DNA sample for comparison. The fragments analysis mix shown below is prepared by the client and submitted to the facility.

  Fragment analysis results are sent to the client using the email provided on the submission form with the standard (.fsa) extension.

  If a sample fails to produce any readable fragments due to a technical error on our part, it will be rerun automatically using the original sample at no additional charge.

• Ion Torrent NGS

  This is a service for clients who perform their own PCR, DNA or RNA extraction and request that our facility perform library prep, emulsion PCR, quality control checks and high throughput sequencing. The 318 chips have 11 million wells and typically 60-70% of the wells load successfully. The Ion PGM™ Hi-Q™ View Sequencing Kit contains reagents and consumables for robust and highly accurate sequencing of 200 and 400 base pair libraries using the Ion OneTouch™ 2 System combined with the Ion PGM™ System. The cost is $1,500.00 per sample and $200.00 for each additional barcoded sample up to 8 barcodes. All samples submitted must be accompanied by a completed Sample Submission Form. (Five working days turnaround)

  DNA samples must be submitted in 1.5 ml Eppendorf tubes with a 10-20 uL volume of DNA no less than 20 ng/uL. This is all that is needed for the facility to start processing DNA samples.

  RNA samples must be submitted in 1.5 ml Eppendorf tubes with a 10-20 uL volume of RNA no less than 20 ng/uL.

  RNA samples must first be determined on the BioAnalyzer to assess the quality of the Sample provided. An RNA integrity number (RIN) greater than 7 or higher is recommended as acceptable starting material for next generation sequencing on the Ion Torrent. Each Agilent RNA 6000 Pico Chip run on the BioAnalyzer cost $48.00 and has 11 wells for 11 RNA samples. Once the RNA quality is acceptable samples from that point forward by the facility.

  Ion Torrent sample results are sent to the client within five working days using the email provided on the submission form. Sample files will have the standard FastQ format.