Sanger DNA SEQUENCING

Service

Up to 1100 bp sequencing resolution

DNA Sequencing from plasmids or PCR products.
We also propose direct DNA sequencing on colony or bacterial culture without necessity of plasmid preparation.

Personalized follow-up, expertise and advice for “difficult” matrices.

How to ask for a sequencing service?

Preparation of templates to be sequenced:

You have the choice of the purification technique for plasmids preparation, i. e. the classic alkaline lyse (phenolic extraction) or use of kits. In you use a commercial kit, do not use the elution buffer provided in the kit and use milliQ H2O.
The quality and the quantity of the templates must be checked on agarose gel using the concentration marker available on the platform (pGEM at 200 ng / µL). The optical density of your samples must be measured : OD at 260 nm and DO260nm/DO280nm.

For samples in strips of 0,2 ml (up to 48 samples)

  1. The tubes have to contain 17 µL of sample (DNA template + primer), prepared according to the conditions recommended below;
  2. The tubes of the strips have to be numbered (at least the first and last tubes of your strip);
  3. Write your identifiers on the caps following this syntax: number of your laboratory – user initials – number of samples (example: 406-AM-10);
  4. Fill out the form for sequencing in strips by giving short names to your samples;
  5. Don’t use the following caracters / , ; : ‘ @ # ( ) in your sample names;
  6. Identify the form according to the following syntax: MMDDYYYYY-number of y our laboratory-initials of the user-number of samples (example: 05282017-406-AM-10);
  7. Fill out and send the form to aeg@ibmp-cnrs.unistra.fr with the object ” sequencing request in strip “.

 For strips with 17 µL (template + primer)/tube:

TEMPLATES QUANTITIES PRIMERS
Plasmids 600 ng > 20 pmoles

 

17 mer < taille < 25 mer

Tm ≥ 50 ° C
PCR products 100 ng

For samples in 96 wells microplates (for more than 48 samples)

  1. Please only use microplates compatibles with the Sanger sequencing available at the platform,
  2. The wells of the plates have to contain 17 µL of sample (DNA template + primer), prepared according to the conditions recommended below;
  3. Fill the plate column by column, from top to bottom, and from the left to the right (1st well: A01, 8th well: H01 … last well: H12);
  4. Identify the microplate with your name and the date of the sequencing request;
  5. Fill out the form for Sanger sequencing in microplate by giving short names to your samples;
  6. Don’t use the following caracters / , ; : ‘ @ # ( ) for your sample names;
  7. Identify the form according to the following syntax: MMDDYYYYY-number of your laboratory-initials of the user-number of samples (example: 05282017-406-AM-56);
  8. Fill out and send the form to the platform’s e-mail address aeg@ibmp-cnrs.unistra.fr with the object ” sequencing request in microplate “;
  9. Put your samples in the freezer on the platform.

For microplates with 17 µL (template + primer)/well:

TEMPLATES QUANTITIES PRIMERS
Plasmids 600 ng > 20 pmoles

 

17 mer < taille < 25 mer

Tm ≥ 50 ° C
PCR products 100 ng

Direct sequencing on colonies, in tubes or microplates

Choose an isolated colony or use a culture to have enough bacteria.

Strain type

 Quantity
High copy plasmid 1 big colony or 70µl culture
Low copy plasmid 200 µl culture

To prepare your microplate or your tubes, use this protocol.

Universal primers available on the platform

Some primers (AEG primers) are available in the freezer of the platform at a 100µM concentration. Just take an aliquot and use your dilutions. Please bring the primers back to the platform as soon as possible.

Your sequencing results

Sequencing results (Sanger / NGS) are currently shared via Offshore:  smb://offshore.ibmp.unistra.fr/sequencage$/

We remind you that Offshore must not be used as a storage space.

Your data will be available 1 mounth and then automatically erased. It is your responsibility to download and store these data.

You will find 2 files:

  • .seq file that contains the nucleotide sequence as a text. You can open it with word, wordpad, texedit, MacVector….
  • .ab1 file that contains your chromatogram.

You can open it with: