• Standard Procedures
• Sample Submission Form

Contact the MIC sequencing manager for information regarding fragment analysis.

Instructions for Submitting Samples for SANGER Sequencing

Fees:
$2.92 per sample template*

*Rate charged to  RI-INBRE users for FY2022.

URI Users:

Use the RI-INBRE MIC as the vendor (PeopleSoft Vendor ID = URI_INBRGS).

Sample Preparation:

Follow our Sample Preparation Guidelines in order to optimize your template and primer for the Applied Biosystems BigDye® Terminator v3.1 chemistry. Template purification and quantification are the most critical factors in obtaining good sequence data. If you are unsure of the quality of your DNA, please check on an agarose gel before submitting the sample. We are not responsible for poor sequences due to impure templates.

Sample Submission:
Fill out a Submission Form to identify your samples and template type. Please note, that as of July 1, 2021, you must obtain the new Submission Form from the MIC! To facilitate billing and file distribution, please provide all the information at the bottom of the form: Name, Principle Investigator, Department / Institution, PO Number and email address.

Target amounts for dsDNA templates as of July 1, 2021:

• PCR products: 1.25 ng DNA per 100 bases per reaction x 2 reactions
• Plasmids: 200 ng DNA per reaction x 2 reactions

Primer amount:

• Use one primer only; either forward or reverse, but not both!
• 1 ul of 3.2 µM primer x 2 reactions

Single sample volume:

• 6 µl per reaction; add template plus one primer in the amounts above to MB grade water.

To facilitate pipetting, submit your sample in duplicate (in the same tube) with a total volume of 12 µl.

Submit your template and primer (combined) in 8-tube strip-tube(s) (capped) or a 96-well plate (capped or sealed). If submitting less than 8 samples, you may submit them in a 0.5 or 1.5 ml tube.

The Sanger sequencing Big Dye reaction is very sensitive to DNA template concentrations. If the calculated volume of template to be added to the duplicate reaction is < 1 µl, please either dilute and requantify the template, or submit triplicate or quadruplicate reactions, making sure to also increase the amount of primer added.

Labeling Samples:
Please identify your samples using the following code: Your initials followed by the number 1 to 9999 (i.e.: PJ1, PJ2, etc.). You should increment the number with each submission. This code facilitates instrument plate setup and data file management.

Sample Analysis:

Sequencing on the 3500xl genetic analyzer is routinely conducted using POP7 polymer, a 50 cm long capillary array and the KB Basecaller software. These conditions normally produce high quality sequence that extends to 800-1,000 bases.

PCR products less than 900 bp in length will be analyzed using an analytical protocol that looks for the end of the raw data. Please identify your PCR product and its size on the Submission Form so this protocol may be specified during instrument setup.

ALL sequence data will be examined for quality and content. Please note that sequence data are NOT trimmed or edited!

Results:

Sequence data will be exported in the standard Applied Biosystems formats, ab1 and seq (ABI and FASTA text only sequence), and distributed by email upon completion of the run. Please be sure to clearly write your email address on the submission form.

Schedule:

Sanger sequencing is performed weekly on Tuesdays and Thursdays.  If you have any questions or problems, call the MIC sequencing manager at 874-5919.

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Sample Preparation Guidelines For SANGER Sequencing

In order to optimize your template and primer for the Applied Biosystems BigDye® Terminator v3.1 chemistry, please consider the following:

DNA Template:

Template preparation is the most critical factor in obtaining good sequencing data. If you are unsure of the quality of your DNA, please check it on an agarose gel or with the NanoDrop before submitting the sample.

Purification – Plasmids and PCR products should be purified before submission. The template should have an OD260/OD280 ratio close to 1.8. Make sure there is NO RNA in the sample.

Quantification – The quantity of template to be used in the sequencing reaction is given below. Quantification can be achieved by spectrophotometric determination (Nanodrop) or by commercial assay kits such as Qubit.

Resuspension & Dilution – Use sterile deionized water (molecular biology grade) or 10 mM Tris-HCL (pH 8.5). EDTA should NOT be present as it can inhibit the sequencing reaction.

Target amounts for for dsDNA templates: 

• 2.5 ng DNA / 100 bases per reaction for PCR products
• 300-500 ng DNA per reaction for plasmids

Primer:

The sequencing primer should be used at a concentration of 5.0 pmol per reaction.

Primers can be ordered from a number of different vendors. Please follow the suggestions below in order to optimize your primer.

Selecting Sequencing Primers – The choice of sequencing primer sequence, method of primer synthesis, and approach to primer purification can have a significant effect on the quality of the sequencing data obtained in dye terminator cycle sequencing reactions with this kit. These decisions are particularly important when sequencing is done on real-time detection systems where signal strength is critical. Some of the recommendations given here are based on information that is general knowledge, while others are based on practical experience gained by Applied Biosystems scientists.

Optimizing Primer Selection – The following recommendations are provided to help optimize primer selection:

• Primers should be at least 18 bases long to ensure good hybridization.
• Avoid runs of an identical nucleotide, especially guanine, where runs of four or more Gs should be avoided.
• Keep the G-C content in the range 30-80%.
• For cycle sequencing, primers with melting temperatures (Tm) above 45 °C produce better results than primers with lower Tm.
• For primers with a G-C content less than 50%, it may be necessary to extend the primer sequence beyond 18 bases to keep the Tm >45 °C.
• Use of primers longer than 18 bases also minimizes the chance of having a secondary hybridization site on the target DNA.
• Avoid primers that have secondary structure or that can hybridize to form dimers.
• Several computer programs for primer selection are available. They can be useful in identifying potential secondary structure problems and determining if a secondary hybridization site exists on the target DNA. We suggest that you consider using Lasergene’s Primer Select for this task (see Lasergene under the Services menu)

The BigDye® Terminator v3.1 Cycle Sequencing Kit – Protocol (©2002 Applied Biosystems) also contains the following information which may help in your sample preparation:

DNA Quality:

Poor Template Quality
Poor template quality is the most common cause of sequencing problems. The following are characteristics of poor quality templates:

• Noisy data or peaks under peaks
• No usable sequence data
• Weak signal

Always follow recommended procedures to prepare templates.

Contamination
Potential contaminants include:

• Proteins
• RNA
• Chromosomal DNA
• Excess PCR primers, dNTPs, enzyme, and buffer components (from a PCR amplification used to generate the sequencing template)
• Residual salts
• Residual organic chemicals such as phenol, chloroform, and ethanol
• Residual detergents

Determining DNA Quality
The following methods can be used to examine DNA quality:

• Agarose gel electrophoresis. Purified DNA should run as a single band on an agarose gel. If multiple bands are present for PCR products, the same is unlikely to sequence well.
  Note: Uncut plasmid DNA can run as three bands: supercoiled, nicked, and linear.
• Spectrophotometry. The A260/A280 ratio should be 1.7 to 1.9. Smaller ratios usually indicate contamination by protein or organic chemicals. Agarose gels reveal the presence of contaminating DNAs and RNAs, but not proteins. Spectrophotometry can indicate the presence of protein contamination, but not DNA and RNA contamination.