Sanger sequencing FAQ

Last updated 21st August 2020

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 Edinburgh Genomics 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 groups.

How do I submit samples for genotyping?

Please click here to view the new guidelines.

We run ABI GeneMapper analyses on full plates only.

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.

How do I submit samples for sequencing?

Please click here to view the new guidelines.

If you have already performed the sequencing reaction:

  • Samples must be 10 µl volumes.
  • The samples should be clearly labelled with your name and the samples numbered clearly and uniquely.
  • Do not clean your samples up: we will clean them up prior to running on the 3730XL.

If you need us to carry out the sequencing reaction, supply clean DNA and the sequencing primer.

  • The primer and template should be premixed to a volume of 6 µl.

For example you provide:

Clean DNA

5 µl

Primer (6.4 pmole/µl)

1 µl

 

How much does it cost?

The following charges apply to the Edinburgh academic community as well as NERC/NBAF projects, as of August 19, 2020. For all other charges, please contact us.

Fragment Analysis (microsatellites, AFLPs)

We only run fragment analysis on full plates (96 samples).

Full plate

£100.00

 

Sequence Analysis

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

Run only

£198 per plate

Sequencing reaction and run

£363 per plate

Prices quoted are exclusive of VAT. Please read our VAT policy (contained within our FAQs) 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 an Edinburgh Genomics 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:

DNA Type

Recommendation

100-200bp PCR product

1.5-3ng

200-500bp PCR product

4.5-10ng

500-1000bp PCR product

7.5-20ng

1000-2000bp PCR product

15-40ng

>2000bp PCR product

60-100ng

Single-stranded DNA from M13 phage vectors

50-100ng

Double Stranded DNA from standard plasmid vectors

300-400ng

Double Stranded DNA from cosmid or BAC vectors

500-1000ng

 

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 1µl of 6.4 µM primer solution for ‘reaction required’ samples.

For larger multi-plate 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 up to 1 week for most submissions. 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?

Edinburgh Genomics 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.