The Pennington Biomedical Research Center Genomics Core Facility operates
two Applied Biosystems 3130XL sequencers.
Large and small sequencing projects are easily accommodated for internal
and external customers.
Customers may send their template for sequencing reaction and electrophoresis
service or may send Big Dye Terminator
sequencing reactions for electrophoresis only.
Turn around time is normally 24 hours for electrophoresis service and 2 days
for sequencing reactions plus electrophoresis.
Turn around time may be extended in the case of personnel shortage, equipment
failure, or high volume.
Sequencing results are placed on a server for download from this web site to your
computer. Results are provided in electropherogram and .Seq file formats.
Printouts are available on request at an additional charge.
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Academic Price List:
| Sample Type | Unit Cost |
|---|
| Electrophoresis only | $3.00 |
| Fragment Analysis | $3.00 |
| High-Throughput Sequencing | $3.75 |
| Reactions and Electrophoresis | $12.50 |
Non-Academic Price List:
| Sample Type | Unit Cost |
|---|
| Electrophoresis only | $5.00 |
| Fragment Analysis | $5.00 |
| High-Throughput Sequencing | $5.00 |
| Reactions and Electrophoresis | $15.00 |
Additional charges
- Expedited service at a cost of $50 per hour for sequencing outside of standard work hours.
- Print request at an additional cost of $1 per sample.
- Big Dye Terminator Dye Removal at an additional cost of $2 per sample.
Policy regarding failed sequences
- Equipment failure, GCF reagent failure, or operator error causing sequence
failure - sequence will be rerun at no additional charge
- All reruns requested by the customer of the same template and primer will be charged
for the first and the second run if the second run is unsuccessful. Changing the template
and/or primer constitutes a new sample submission for which you will be charged.
To request a rerun, provided enough sample and primer remain, the customer must
re-enter the sample information on this web site and provide the new plate number to
GCF
.
- Failure of sequencing reactions performed outside of GCF will be assumed to be the responsibility of the performing lab except in the event of an equipment malfunction.
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Four options are available for submitting samples to the Genomics Core for sequencing:
- Reaction & Electrophoresis service: Template and primer are submitted
separately. The Genomics Core will set up the sequencing reactions, clean the
reactions of excess Big Dye Terminator dyes, and run the reactions on the sequencer.
This full service option is suggested when you have less than 15 samples to sequence.
Template and primer requirements for Reaction & Electrophoresis service
- Electrophoresis only service: Your laboratory runs the sequencing reactions
and removes excess dye. The Genomics Core runs your reactions on the sequencer.
Requirements for Electrophoresis only service
- High-Throughput sequencing service: Your sample and primer are submitted
as a mixture for each sequencing reaction in a 96-well sequencer compatible plate.
The Genomics Core adds the sequencing reagents, cycles the plate, cleans the excess
dye from the reactions and runs the plate on the sequencer.
Requirements for High-Throughput sequencing service
- Fragment Analysis: You submit fluorescent-labeled PCR fragments for analysis.
Requirements for Fragment Analysis
Template and primer requirements for Reaction & Electrophoresis service
| DNA Template | Template Concentration | Primer Concentration |
| Plasmid (Double Stranded DNA) | 25 ng/uL | 10 micromolar |
| PCR Product - 100-200 bp | 1.5 ng/uL | 10 micromolar |
| PCR Product - 200-500 bp | 5 ng/uL | 10 micromolar |
| PCR Product - 500-1000 bp | 10 ng/uL | 10 micromolar |
| PCR Product - 1000-2000 bp | 20 ng/uL | 10 micromolar |
| PCR Product - >2000 bp | 25 ng/uL | 10 micromolar |
| Single Stranded DNA | 5 ng/uL | 10 micromolar |
| Cosmids, BAC | 200 ng/ul | 10 micromolar |
- Samples and primer must be dissolved in distilled water.
- Do not mix the template and primer.
- Provide 10 ul of the template for each reaction desired.
One template/primer set equals one sequencing reaction. One template sequenced with
two primers equals two sequencing reactions, etc.
- If you are submitting 20 or more samples, the template must be provided in a standard
half-skirted 96 well plate. The templates should be placed in column order as listed
on the sequence submission sheet. The plate must be labeled with the plate name.
- Less than 20 samples may be submitted in tubes with the well number from the submission
form written on top and the plate name written on the side of the tube.
Primers
- Primers provided by the Genomics Core Facility:
| M13 Universal primer | 5’-GTA AAA CGA CGG CCA GT-3’ |
| M13 Reverse primer | 5’-AAC AGC TAT GAC CAT G-3’ |
| SP6 Sequencing primer | 5’-GAT TTA GGT GAC ACT ATA G-3’ |
| T3 Sequencing primer | 5’-ATT AAC CCT CAC TAA AG-3’ |
| T7 Sequencing primer | 5’-AAT ACG ACT CAC TAT AG-3’ |
- Custom primer provided by customer must be 10 micromolar concentration.
Provide 2 microliters per sequencing reaction plus 5 microliters excess.
Requirements for Electrophoresis only service
- A sequencing standard reaction must be submitted with all Big Dye Terminator
sequencing reactions. Failure to submit the standard will result in the assumption
that any sequencing failures are the fault of your laboratory. GCF will have no liability.
- If you are submitting 20 or more samples, the template must be provided in a
standard half-skirted 96 well plate. The sequencing samples should be placed in
column order as listed on the sequence submission sheet. The plate must be labeled
with the plate name.
- Less than 20 samples may be submitted in tubes with the well number from the
submission form written on top and the plate name written
on the side of the tube.
Requirements for High-Throughput sequencing service
| DNA Template | Template Volume and Concentration | Primer Volume and Concentration |
| Plasmid (Double Stranded DNA) | 1 microliter @ 25 ng/uL | 1 microliter@1 micromolar |
| PCR Product - 100-200 bp | 1 microliter @ 1.5 ng/uL | 1 microliter@1 micromolar |
| PCR Product - 200-500 bp | 1 microliter @ 5 ng/uL | 1 microliter@1 micromolar |
| PCR Product - 500-1000 bp | 1 microliter @ 10 ng/uL | 1 microliter@1 micromolar |
| PCR Product - 1000-2000 bp | 1 microliter @ 20 ng/uL | 1 microliter@1 micromolar |
| PCR Product - >2000 bp | 1 microliter @ 25 ng/uL | 1 microliter@1 micromolar |
| Single Stranded DNA | 1 microliter @ 5 ng/uL | 1 microliter@1 micromolar |
Mix 1 microliter of the template and 1 microliter of the primer at the
appropriate concentration listed in the table above into a single well
of a half-skirted 96-well PCR plate (Life Technologies part number N8010560
or equivalent). Samples should be placed in column order on the plate
matching the sequence submission well numbers on the sequence submission sheet.
Seal the plate securely.
Samples submitted for the High-Throughput service must meet the following requirements:
- The minimum order is 15 samples.
- Samples are submitted as a mixture of template and primer in the amounts specified above.
- The Genomics Core does not provide primers for High-Throughput sequencing.
- Samples are submitted in a half-skirted 96 well plate compatible with the 3130XL sequencer.
- Samples will not be rerun unless there is a mechanical failure of the instrument.
Requirements for Fragment Analysis
-
If you are submitting 20 or more samples, the template must
be provided in a standard half-skirted 96 well plate. The sequencing samples
should be placed in column order as listed on the sequence submission sheet.
The plate must be labeled with the plate name.
-
Less than 20 samples may be submitted in tubes with the well
number from the submission form written on top and the plate name written
on the side of the tube.
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- Generate PCR fragments using one dye-labeled primer and one standard primer.
Dyes that may be used are: 6FAM, NED, VIC, and PET.
- Clean up the PCR reaction to remove excess dNTP’s and primers.
- The appropriate concentration of your fragments must be determined empirically in
order to find the correct signal strength during the run. This may require running
several dilutions of the PCR product on the sequencer in order to determine the best
dilution.
- After dilution, add the appropriately sized fragment analysis standard to each sample.
The dye, LIZ, is normally used as the label for the standard.
- Fragment analysis samples may be multiplexed using different dyes and/or different
sized PCR fragments to increase throughput. This may be done by optimizing a multiplexed
PCR reaction or by running individual reactions and combining the products before submitting
the samples to the Genomics Core Facility. Multiplexing a PCR reaction will reduce the
concentration of each PCR product in the overall mixture. Please keep this in mind when
selecting the injection time for your run.
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-
Login to the GCF web site (New customers must register first) by going
here.
- Submit new samples.
- Submit the plate to GCF.
- Package and label your samples:
- the well number assigned to the sample should go on the top
- the plate number should go on the side
Remember that 20 or more samples must be in a half-skirted 96-well plate.
- Deliver Samples:
Samples delivered on Friday will not be processed until the following week.
- Pennington employees may deliver samples to the top shelf box in the freezer
located in room L2012. Leave associated paperwork in the folder on the door of the freezer.
- Customers outside of Pennington may either
- Deliver samples to the Administration building (faces Perkins Road) reception
desk between 8 AM to 4:45 PM Monday through Thursday and 8 AM to 4 PM on Friday.
OR
Samples may also be shipped by overnight shipment if packaged on ice or with
cold packs. Overnight shipments must be scheduled to arrive during business hours.
The shipping address is:
Genomics Core Facility
Pennington Biomedical Research Center
6400 Perkins Road
Baton Rouge, LA 70808
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- Plasmid
The host strain and plasmid used may affect the quality of the sequence. Use a purification method which specifically states that it produces sequencing quality DNA. Contaminates cause excess background noise and/or weak signal which may result in unreadable DNA sequence.
- Recommended purification methods
- Qiagen mini kits
- Cesium Chloride centrifugation
- Modified alkaline lysis/PEG method.
- PCR
Excess primers, PCR buffers, and dNTP’s must be removed after amplification. The PCR amplification must be a single band. PCR reactions which are not clean (non-specific bands, primer dimer) will not sequence well.
- Clean-up methods for single band PCR
- For PCR products with non-specific products
- QiaexII Gel Extraction kit
- Qiaquick Gel Extraction kit
- Single Stranded DNA
- Qiagen Qiaprep M13 kit
- Thermomax procedure
- PEG precipitation and phenol extraction.
- BAC’s
- alkaline lysis
- Cesium chloride centrifugation
- Qiagen Large Construct kit
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- Primer Design Software
- Primer design rules and tips
- Length of 18-20 base pairs if possible
- Avoid runs of a single base, especially 3 or more G’s or C’s
- Melt temperature above 50 C generally works better. The melt temperature of a primer may be estimated by:
Tm=(Number of A + T bases) X 2º C + (Number of G + C bases) X 4º C
- G/C content 40-60%, lengthen primer if needed to maintain a melt temperature of 50º C
- GC clamp on 3’ end
- No hairpins or sequences which may lead to primer-dimer
- Verify that the primer binds to a unique sequence if possible
- No degenerate primers
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Excess dye terminators must be removed from sequencing reactions prior to submission.
- Applied Biosystems Big Dye XTerminator
| Part Number | Product Name |
|---|
| 4376486 | BigDye® XTerminator™ Purification Kit - 2 mL (~100 - 20 µL reactions) |
| 4376487 | BigDye® XTerminator™ Purification Kit - 20 mL (~1,000 - 20 µL reactions) |
| 4376484 | BigDye® XTerminator™ Purification Kit - 50 mL (~2,500 - 20 µL reactions) |
| 4376485 | BigDye® XTerminator™ Purification Kit – 800 mL |
- Edge Biosystems
| Part Number | Product Name |
|---|
| 42453 | Performa DTR Gel Filtration Cartridges |
| 91751 | Performa DTR 96 well standard plates |
- Ethanol or Isopropanol precipitation: Less dye removal. This method will result in dye peaks obscuring the first 10-20 bases.
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Web Resources for Sequence Viewing
Sequence Viewing and Multiple Alignment Software
To increase the read length of your electropherograms computationally
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More Troubleshooting
- Normal sequence
- low or no background peaks
- limited excess dye peaks
- plasmid read length >700 base pairs with <2% error or ambiguity
- signal strength 50-800
- Sequencing problems
- no readable sequence
- template
- concentration is incorrect
- template contains contaminants inhibitory to the sequencing reaction
- mutation in the primer binding site
- PCR reaction not cleaned before sequencing
- primer
- primer does not bind to the template due to mutation, secondary structure, etc
- primer-dimer or hairpin formation
- incorrect primer concentration
- incorrect primer melt temperature
- High level of noise, sequence difficult to read accurately
- Low signal strength > 50 due to low template concentration
- Dirty template
- High GC content
- Overlapping sequences with good peaks on both sequences
- Primer has primed the sequence in more than one location
- Two PCR fragments present and sequenced. One fragment may be shorter than the other resulting in clean sequence after the shorter fragment sequence ends.
- PCR reaction primers not completely removed so PCR primers sequence from both ends of fragment.
- Sequence starts with good peaks which rapidly drop
- salt in the template or sequencing reaction
- secondary structure in template
- Drop off of sequencing following homopolymer regions: Enzyme “skips” through region. Primer must be designed on opposite strand or in new area in an attempt to sequence through the homopolymer.
- Pull-up minor peaks under sequence peaks: Too much template or Big Dye Terminator in the sequencing reaction
- Excess dye peaks around 70 base pairs, 90 base pairs, 210 base pairs, 240 base pairs: unincorporated dye terminators not removed from the sequencing reaction during cleanup
- Minor N-1 peaks in addition to major peaks: primer degraded due to excessive freeze thaw or poor manufacture
- Clean sequence which becomes double: hairpin loop or secondary structure, insertion or deletion
- Rolling hills: bubble in capillary
- Peaks start late and are broad with loss of resolution
- Incorrect concentration value leading to too much template in the sequencing reaction
- Overloaded plasmid prep column
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Primer Design
Web Resources for Sequence Viewing
Sequence Viewing and Multiple Alignment Software
To increase the read length of your electropherograms computationally
To Unzip Sequence Electropherogram files
Troubleshooting electropherograms
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