genepool

Next Generation Sequencing and Bioinformatics
at the University of Edinburgh


Sanger Sequencing - FAQ

This collection of frequently asked questions (FAQ) provides answers to some of the most common queries we receive about Sanger sequencing. Please contact us if your question is not covered here.

Who does the GenePool offer Sanger sequencing to?

How do I submit samples for genotyping?

How do I submit samples for sequencing?

Can you help with large sequencing projects?

Where should I take my samples?

How much does it cost?

Do I need to pay VAT?

How much DNA do I need?

How much primer do I need?

I have submitted samples. When will I get my results?

How do I access my results?

How do I view my results?

I have a NERC grant -how should I apply for sequencing?

I have a NERC grant and was awarded access to the sequencing facility. How do I gain access to my allocation?

Why didn’t my sequences work?

 

Who does the GenePool offer Sanger sequencing to?

We offer Sanger sequencing to:

*Edinburgh University researchers

*Researchers in Institutes and other research centres in the Edinburgh area

*Researchers from throughout the UK whose science falls within the NERC science area. Researchers with projects falling in this category are invited to apply for access to our Sanger sequencing facility through the NERC Biomolecular Analysis Facility (NBAF).

We do not usually offer access to our Sanger technology to groups outside these constituencies.

How do I submit samples for genotyping?

Our sample submission procedures have changed. Please click here to view the new guidelines.

We run ABI GeneMapper analyses on either 48 or 96 samples at a time.

If you want to run GeneMapper plates, please come and see us first and we will provide you with barcoded plates and Hi-Di formamide.

Primers must be labelled with the ABI G5 dye set: 6-FAM, VIC, NED, PET.

Samples must be brought in the plates provided in a volume of 10 µl.

For full plates, all 96 wells should be filled - blank wells can be filled with 10 µl of Hi-Di.

For half-plates samples should be placed in alternate “odd” lanes (e.g. 1,3,5,7,9,11). All 48 wells to be processed should contain 10 µl of liquid but the “even” lanes can be left empty.

Plates must be brought to us in Ashworth Laboratory 352 before 3 pm.

How do I submit samples for sequencing?

Our sample submission procedures have changed. Please click here to view the new guidelines.

If you have already performed the sequencing reaction:

Samples must be 10 µl volumes and brought in 96 well plates or in 0.2 ml strip tubes. We are sorry that our operating procedures do not allow us to accept any other tube format.

The samples should be clearly labelled with your name and the samples numbered uniquely.

Do not clean your samples up: we will clean them up prior to running on the 3730.

If you need us to carry out the sequencing reaction:

We will sequence your DNA for you, if you supply clean DNA and the sequencing primer.

The primer and template should be premixed to a volume of 6 µl and brought in 0.2 ml strip tubes or 96 well plates.

For example you provide:

Clean DNA
5 µl
Primer (3.2 pmole/µl)
1 µl

Can you help with large sequencing projects?

We are fully equipped to perform larger sequencing projects – from thousands of ESTs to the sequencing of large insert clones. Please contact us to discuss your project. These larger projects are processed and costed per 96 well plate of reactions.

Where should I take my samples?

Ashworth and ISCR users should take their samples to the collection fridge or freezer in Laboratory 352 of Ashworth 3.

Darwin, Rutherford and Swann users should take their samples to the collection fridge on the 5th floor of Darwin.

The collection fridge/freezer will be emptied at 11 am daily.

How much does it cost?

The following charges apply to the Edinburgh academic community as well as NERC/NBAF projects, as of August 01, 2011. For all other charges, please contact us.

Fragment Analysis (microsatellites, AFLPs)

We only run fragment analysis on 48 or 96 samples at a time.

48 samples
£45.00
96 samples
£75.00

Sequence Analysis

We offer a range of sequencing analyses from analysis of sequenced samples (run only) to full template analysis (i.e., PCR, SAP, sequencing reaction, and run).

Run only 
£1.50 per sample
£130.00 per plate
Sequencing reaction & run
£3.75 per sample
£325.00 per plate
Full analysis (plates only)  
not available 
£525.00 per plate

We also offer bioinformatics support and volume discounts for large projects. Please contact us to discuss your needs.

Prices quoted are exclusive of VAT. Please read our VAT policy here.

A Purchase Order must be supplied with each sample submission form by all users outwith the University of Edinburgh (unless you have a NERC/NBAF allocation directly provided to us by NERC).

Do I need to pay VAT?

All our quoted prices are exclusive of VAT. For tax purposes, our Sanger sequencing work is regarded as a non-collaborative research service. Thus VAT will added by default at the applicable rate to all orders charged against funds outside of the University of Edinburgh (i.e. when a purchase order is supplied to us). Please note that orders charged to funds held within the University of Edinburgh are not liable to VAT.

DNA sequencing is classified as supply of services and thus zero-rated VAT certificate for goods will be not be accepted as a proof of VAT exemption. However if you hold a research grant with a GenePool academic, you may apply for VAT relief by providing supporting documentation as justification. Documentation for VAT relief must submitted for each order, at the time of sample submission through our GeneSifter LIMS system.

How much DNA do I need?

We require different amounts of DNA template depending on the type and size of fragment. ABI recommends the following quantities:

For purified PCR products
 
100-200 bp
1-3 ng
200-500 bp
3-10 ng
500-1000 bp
5-20 ng
1000-2000 bp
10-40 ng
> 2000 bp
40-100 ng
For single-stranded DNA from M13 phage vectors
50-100 ng
For double-stranded DNA from standard plasmid vectors
200-500 ng
For double-stranded DNA from cosmid or BAC vectors
0.5-1.0 µg

Important note. We find that DNA concentration as determined by spectrophotometer (e.g. Nanodrop) is highly unreliable. We strongly recommend using a fluorometer (e.g. Invitrogen Qubit) for quantification of DNA samples submitted for Sanger sequencing.

How much primer do I need?

We require 1 µl of 3.2 µM primer solution for ‘reaction required’ samples.

For larger projects, we require primers at 10 µM concentration. We can arrange for synthesis of stocks of primers required for large-scale projects – please ask.

I have submitted samples When will I get my results?

Our turnaround is usually 1 business day for genotyping samples, 1-2 business days for ‘sequencer only’ samples, and 2-3 business days for ‘reaction required’ samples. For larger projects, we will discuss turnaround times with you before you submit your samples.

How do I access my results?

Results can be downloaded from within our LIMS. You will be sent a link via e-mail when the results are ready.

How do I view my results?

You can use commercial software or try one of these freeware programs:

PC Chromas
MAC 4Peaks
PC & MAC FinchTV
PC & MAC MEGA

I have a NERC grant -how should I apply for sequencing?

The GenePool is a collaborating node in the Natural Environment Research Council’s Biomolecular Analysis Facility (NBAF) and researchers with projects falling within the NERC science area can apply for access to the facility.

NERC-NBAF access is awarded competitively, either through grant awards from NERC through the usual channels (when a statement of request for access to the facility needs to be made in the application form, and you should have discussed your sequencing needs with us before you submit) or through direct application to the NERC-NBAF Scientific Steering Committee. For direct applications, you should also contact us to discuss your needs before submitting the application.

Please note that NBAF also offers molecular genetic/population genetic (microsatellite and SNP genotyping), microarray and high throughput Roche 454 and Illumina SOLEXA sequencing services along with bioinformatics support. See check the NBAF website for details of what NBAF offers and how to apply for access.

I have a NERC grant and was awarded access to the sequencing facility. How do I gain access to my allocation?

Even though you have been awarded an allocation with your grant you still need to apply to the NBAF committee to gain access to your allocation. You will be given a unique NBAF number that you need to quote when you submit your samples. Application forms can be found here.

Why didn’t my sequences work?

Sequencing reactions can fail for any number of reasons. Please consult our troubleshooting guide or contact us and we will work with you to improve your results.


The GenePool has core support from: GenePool admin site
edinburgh
The University of Edinburgh
School of Biological Sciences
The Darwin Trust
of Edinburgh
darwin
mrc
nerc
bbsrc
sulsa

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This is version 2.2 of the GenePool website (July 2009).