Sanger Sequencing Guidelines
Submission Guidelines
Courier Service
Sample drop boxes are located in Partners II, Partners III, and the CVM Research Building for your convenience. We offer same-day service (by 5 pm) for Gold and Bronze sequencing, and next-day service for Platinum sequencing. Submissions requesting Courier Pickup MUST be received by 9:30 am the day that you are requesting sample collection. Your samples MUST be in the dropbox by 10:00 am for the pickup. Samples submitted after this time will be collected on the next business day.
Plate Sequencing
All plates submitted must be 96-well semi skirted PCR Plates (available for purchase at the GSL). Use of one of the following plate types is required for plate submission:
- 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 Tube Sequencing
- For local drop-off, users may submit samples in 1.7mL tubes in a tube rack labeled with a piece of tape with the Customer Name, PI Name, and Date. If submitting via one of our Courier Dropboxes (Partners II, Partners III, CVM Research Building) or by mail/FedEx, please ensure your sample tubes are sufficiently wrapped or boxed to ensure safe transport to our facility.
- Label tubes legibly on the top of the cap with initials and a number (Ex. GSL-1).
- When submitting 8 or more single tubes for sequencing, users should submit their samples in an 8-well PCR strip tube or 96 well plate (uncapping/recapping many individual tubes slows down our ability to process).
- Make sure samples are in the same order in the rack as on your sample sheet.
- If dropping off samples directly at the GSL, samples and submissions sheets must be received before 11:00 am for same day service (Gold and Bronze), or next-day service (Platinum). Late submissions will be run the following business day.
- Please do not use any unusual character names as sample names. The only characters our ABI 3730 software will accept are letters, numbers, and underscores (no slashes, dashes, spaces, or other punctuation marks).
Platinum Sequencing
Note: Only Gold and Bronze Sequencing services are available for Next-Day data delivery. Platinum sequencing requires a two-day delivery window for the extra clean-up procedure.
WARNING! – For Platinum samples, DO NOT add primers to the same tube as your DNA as Exosap cleanup is part of the service. Any sequencing primer added to the DNA will be degraded by this process. Please provide your sequencing primers in separate 1.7 mL microfuge tubes or 8-well strip tubes.
PCR Clean-up through Capillary 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 |
|---|---|---|
| 100-200bp | 1.3-4 ng/uL | 8-24 ng/6ul |
| 200-500bp | 4-10 ng/uL | 24-80 ng/6uL |
| 500-1000bp | 6.7-26.7 ng/uL | 40-160 ng/6ul |
| 1000-2000bp | 13.3-53.3 ng/uL | 80-320 ng/6uL |
| >2000bp | 53.3-133.3 ng/uL | 320-800 ng/6uL |
| Single Stranded | 66.7-133.3 ng/uL | 400-800 ng/6uL |
| Double Stranded | 266.7-666.6 ng/uL | 1.6u20134 ug/6uL |
| Cosmid, BAC | 0.67-1.3 ug/uL | 4-8 ug/6uL |
Gold Sequencing
Cycle Sequencing Reaction Set-up through Capillary Run
When sequencing at the Gold level, please submit your samples with 6.4 pmols of primer (either forward or reverse). 6.4pmols of primer is generally about 2 ul of 5 uM primer or 1 ul of 10 uM primer added to each well. DO NOT put both the forward and reverse primer in the same well. Please indicate clearly if you would like the GSL to add one of 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 the primer, the final volume should be 12μl. This provides us with enough material to do 2 reactions in the event that 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.
| Template PCR Product | Quantity |
|---|---|
| 100-200bp | 2-6ng |
| 200-500bp | 6-20ng |
| 500-1000bp | 10-40ng |
| 1000-2000bp | 20-80ng |
| >2000bp | 80-200ng |
| Single Stranded | 100-200ng |
| Double Stranded | 400-1000ng |
| Cosmid, BAC | 1-2.0 ug |
Bronze Sequencing
Cycle Sequencing Reaction Clean-up through Capillary Run
When sequencing at the Bronze level, users are responsible for completing their own Dye terminator sequencing reactions. Following submission of the plate, the GSL will perform the cycle-sequencing reaction clean-up and capillary run. Training can be provided and users can have access to the GSL for these procedures. Applied Biosystems Big Dye 3.1 is required for all sequencing reactions and can be purchased at the GSL. This mixture contains the Taq, 2.5X buffer, Mg 2+, dNTPs and dye-labeled ddNTPs. Simply add your primer (forward or reverse). Information about the reaction set-up and clean-up protocol is below:
Sequencing Steps
- Generation of Template (single-stranded, double stranded, plasmid, BAC, PCR products) preparation via commercial kits, phenol extraction, plasmid prep, etc.
- Purification of product – removal of primers, dNTPs, and other contaminants with ethanol precipitation, commercial kits, etc.
- Quantify DNA using agarose gel electrophoresis, spectrometry, etc.
- Amplification via cycle sequencing with Big Dye (purchase at GSL), primer, and template.
- Clean-up of cycle sequencing samples (done at GSL)
- Submit plate and plate record
- 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 Pricing page)
- Sequencing Buffer: First tube free with purchase of BigDye at GSL
- Water
10μL Final Volume
| Template PCR Product | Quantity |
|---|---|
| 100-200bp | 1-3ng |
| 200-500bp | 3-10ng |
| 500-1000bp | 5-20ng |
| 1000-2000bp | 10-40ng |
| >2000bp | 40-100ng |
| Single Stranded | 50-100ng |
| Double Stranded | 200-500ng |
| Cosmid, BAC | 0.5-1.0ug |
| Bacterial Genomic DNA | 2-3ug |
Full reaction (per sample)
| BigDye | 4uln |
| 5x Sequencing Buffer | 2ul |
| Primer (3.2picomoles) | x ul |
| DNA Template | y ul |
| dH20 | z ul |
| Total Volume | 10ul |
1/2 reaction (per sample)
| BigDye | 2uln |
| 5x Sequencing Buffer | 1uln |
| 3.2 picomoles Primer | x uln |
| DNA | y uln |
| dH20 | z uln |
| Final Volume | 10uln |
1/4 reaction (per sample)
| BigDye | 1uln |
| 5x Sequencing Buffer | 1.5uln |
| 3.2 picomoles Primer | x uln |
| DNA | y uln |
| dH20 | z uln |
| Final Volume | 10uln |
Cycle Sequencing
For Single and Double Stranded DNA:
96oC | 1 min | |
| 96oC | 10 sec | |
| 50oC | 5 sec | 25 cycles |
| 64oC | 4 min | |
| 4oC | Hold |