Sanger Sequencing and Genotyping


Sanger Sequencing

Types of Sequencing Services

  • Platinum Sequencing Service – PCR Clean-up, Cycle Sequencing Reaction Set-up and Clean-up, Sequencing Run and Data Distribution
  • Gold Sequencing Service- Cycle Sequencing Reaction Set-up and Clean-up, Sequencing Run and Data Distribution
  • Bronze Sequencing Service – Clean-up Cycle Sequencing Reaction (you do the reactions), Sequencing Run and Data Distribution
  • Single Reaction Sequencing – Available in Gold or Platinum format. To be eligible for the same day service, samples must be dropped off before 12:00pm that day.
  • Microsatellite Analysis/Fingerprinting Plates – Sequencing Run and Data Distribution

Sample Submission Requirements

Plate Sequencing

All plates submitted must be 96-well semi skirted PCR Plates. The following plates are recommended:

  • USA Scientific: Part Number 1402-9200
  • Genesee Scientific: Part Number 27-408

Please Label the Plate with:

  • The name matching your plate record
  • The level of sequencing service you are requesting (Platinum, Gold or Bronze)

Single Reaction Sequencing

  • Submit samples in 1.7mL tubes in a tube rack labeled with a piece of tape with the Customer Name, PI Name, and Date
  • Label tubes legibly on the top of the cap with initials and a number (Ex. GSL-1)
  • Make sure samples are in the same order in the rack as on your sample sheet
  • Drop off samples before 12:00pm for sample day service
  • Email sample submission sheet as an excel document to GSLNCSU@gmail.com before dropping off samples
  • Please do not use any unusual character names in your form (underscores permitted, spaces are not)
  • Example of Sample and Sample Sheets Submission

Submission Forms

Platinum Sequencing (PCR Clean-up through Run)

When submitting plates for PCR clean-up, samples MUST be submitted in 96-well, semi skirted PCR plates with a reaction volume of at least 6uL.  Products are purified using ExoSAP.

Template
ng/ul
Total ng in 6ul
100-200bp1.3-4 ng/uL8-24 ng/6ul
200-500bp4-10 ng/uL24-80 ng/6uL
500-1000bp6.7-26.7 ng/uL40-160 ng/6ul
1000-2000bp13.3-53.3 ng/uL80-320 ng/6uL
>2000bp53.3-133.3 ng/uL320-800 ng/6uL
Single Stranded66.7-133.3 ng/uL400-800 ng/6uL
Double Stranded266.7-666.6 ng/uL1.6–4 ug/6uL
Cosmid, BAC0.67-1.3 ug/uL4-8 ug/6uL

Gold Sequencing (Cycle Sequencing Reaction Set-up through Run)

When sequencing at the Gold level, please submit your samples in a 96 well plate with 6.4pmols of primer (either forward or reverse). 6.4pmols of primer is generally about 2ul of 5uM primer or 1ul of 10uM primer added to each well. Do not put both forward and reverse primer in the same well. Please indicate clearly if you would like the GSL to add on or our primers, T7, SP6, M13F or M13R. If the GSL is adding primer, please bring your sample to a volume of 10ul. If you’ve included primer, the final volume should be 12μl. This provides us with enough material to do 2 reactions in the event a ‘redo’ is needed. Plate record submission and data retrieval are described below. Please label your plate with the exact same name as the plate record you submit online.

Gold Sequencing:

Template PCR Product
Quantity
100-200bp2-6ng
200-500bp6-20ng
500-1000bp10-40ng
1000-2000bp20-80ng
>2000bp80-200ng
Single Stranded100-200ng
Double Stranded400-1000ng
Cosmid, BAC1-2.0 ug

Bronze Sequencing (Cycle Sequencing Reaction Clean-up through Run)

When sequencing at the bronze level, users are responsible for the preparation and clean-up of their samples prior to submission of the plate. Training can be provided and users have access to the GSL for these procedures. Applied Biosystems Big Dye 3.1 must be purchased at the GSL and is required for all sequencing reactions. This mixture contains the Taq, 2.5X buffer, Mg2+, dNTPs and dye-labeled ddNTPs. Simply add your primer (forward or reverse). Information about the reaction set-up and clean-up protocol appears below.

Sequencing Steps

  1. Generation of Template (single-stranded, double stranded, plasmid, BAC, PCR products) preparation via commercial kits, phenol extraction, plasmid prep, etc.
  2. Purification of product- removal of primers, dNTPs, and other contaminants with ethanol precipitation, commercial kits, etc.
  3. Quantitate DNA using agarose electrophoresis, spectrometry, etc.
  4. Amplification via cycle sequencing with Big Dye (purchase at GSL), primer, and template
  5. Clean-up of cycle sequencing samples (done at GSL)
  6. Submit plate and plate record
  7. Data Retrieval

Sequencing Reaction Setup (BRONZE)

  • Template: See table below for quantity
  • Primer :3.2 pmol of Forward OR Reverse
  • BigDye: Purchase at GSL (see Rates page)
  • Sequencing Buffer: First tube free with purchase of BigDye at GSL
  • Water

10μL Final Volume

Bronze Sequencing:

Template PCR Product
Quantity
100-200bp1-3ng
200-500bp3-10ng
500-1000bp5-20ng
1000-2000bp10-40ng
>2000bp40-100ng
Single Stranded50-100ng
Double Stranded200-500ng
Cosmid, BAC0.5-1.0ug
Bacterial Genomic DNA2-3ug

Full reaction per sample

BigDye4ul
Primer (3.2picomoles)x ul
DNA Templatey ul
dH20z ul
Total Volume10ul

1/2 reaction per sample

BigDye2ul
5x Sequencing Buffer1ul
3.2 picomoles Primerx ul
DNAy ul
dH20z ul
Final Volume10ul

1/4 reaction per sample

BigDye1ul
5x Sequencing Buffer1.5ul
3.2 picomoles Primerx ul
DNAy ul
dH20z ul
Final Volume10ul

Cycle Sequencing

For Single and Double Stranded DNA

96oC
1 min
96oC
50oC
64oC
10 sec
5 sec
4 min

25 cycles
4oC
Hold


Genotyping Services

Genotyping (Microsatellite and Fingerprinting Plates)

The GSL only accepts ready-to-load 96-well plates.

Users are responsible for preparation and clean-up of their samples prior to submission of the plate and have access to GSL for these procedures.

We provide training for these steps and then perform the maintenance and loading of the capillary sequencers.  Genemapper Analysis software version 4.0 is available at the GSL and Peak Scanner Software version 1.0 is available online.

All Genotyping plates are processed on an Applied Biosystems 3730xl DNA Analyzer.  Genotyping on the 3730xl has a wide range of applications including AFLP, Microsatellite, SNP analysis, etc.  The 3730xl is optimized for a 5-dye set (Dye set 33 Filter set G5).  The dyes in this set include 6-FAM, VIC, NED, PET, and LIZ size standard.  The 5-dye set has increased multiplexing capabilities.

Genotyping Steps

  1. DNA extraction via commercial kits, phenol extraction etc.
  2. Quantify DNA using agarose electrophoresis, spectrophotometry, etc.
  3. Perform PCR with dye labeled primers
  4. Multiplex by combining PCR products
  5. Set-up plate with PCR products, HiDi Formamide, and size standard
  6. Submit plate and plate record
  7. Data Retrieval

Genotyping Reaction Setup

  • PCR Template: Optimized by user
  • Size Standard: 0.5uL per reaction
  • High Dye Formamide: Amount needed to bring final reaction volume to 10uL

PCR Template

Users are required to optimize the amount of template best for their samples.  If the user desires to multiplex, it is best to run the PCR reactions separately and then combine them in the final plate.  You can have up to 4 different PCR products in one well; one for each of the available dyes.

Size Standard

LIZ-600 is the available size standard for the dye set optimized on the 3730xl sequencer.  Use 0.5uL per reaction.

HiDi Formamide

Users will need to purchase highly de-ionized formamide from Applied Biosystems.  It is important to get this grade of formamide because others will affect the sequencing run.  Use the formamide to bring the final volume of the reaction to 10uL.  Empty wells should be filled with 10uL of water.

Plate Record Submission

You must submit a plate record before your plate can be sequenced. The plate record must match the name that is physically on your plate. The plate name and sample names must follow the rules below:

  • Must be 10 characters or less
  • No spaces
  • No unusual characters; you may use underscores __
  • Name all wells, if needed use blank, empty, water, etc.

To make a plate record:

  • Download the plate submission sheet and list your sample names in 1 single column. The order should go DOWN your plate columns, NOT across. See image below:
  • Save the plate record at a tab delineated text file (.txt)
  • Email the plate record to the GSL at GSLNCSU@gmail.com

Data Retrieval

Data will be emailed from the GSLNCSU@gmail.com account.

If necessary, contact Jenn, Cory, or Julie