Sanger Sequencing

StarSEQ® can only offer the unbelievable price because of its high degree of automation during the whole sequencing process. Please follow the recommended primer and DNA concentration, since these are essential for the success of the sequencing reaction. Unsuccessful reactions will also be billed. Abbreviation and numbering on top of the lid of your sample tubes must be identical to order form!

1. Login.
2. Fill in the order form electronically.
3. Submit the order form.
4. Receive the completed order form as PDF by e-mail and popup window.
5. Print order form and sign it.
6. Label the reaction containers legibly on the lids with an indelible black pen.
7. Please send your samples and primers in 1.5 ml tubes.
8. Protect the reaction containers with a box or something similar and send together with order form in a padded envelope to:

StarSEQ GMBH, Johann-Joachim-Becher-Weg 30a, 55099 Mainz, Germany

For any questions or requests on your samples please contact us with your copmplete personal details, date of received results and the abbreviation of your samples under the following E-mail address:
v-mix@starseq.com

Recommendations:
DNA: dissolved in 10-15 µl water or TrisHCl (10mM), pH 7-8 (NO EDTA!!!). Minimum concentration of your plasmid DNA template must be 100 ng/µl!
Amounts: purified PCR product: 200 bp: >100 ng; 500 bp: >200 ng 1 kb: >400 ng /sequencing reaction
Plasmid DNA: <3,5 kb: 350 ng; 3,5 – 4,5 kb: 400 ng; 4,6 – 5,5 kb: 450 ng; 5,6 – 6,5 kb: 500 ng; 6,6 – 7,5 kb: 550 ng; 7,6 – 10 kb: 600 ng; > 10 kb: 700 ng /sequencing reaction
Cosmid DNA, PACs, BACs: >1 µg /sequencing reaction
Primer: 3 µl of a 10 µMolar primer solution for each rxn, standard primer available. You find the list here. For 48- or 96-well plates only one primer should be used for the whole plate. Or, another pair plate for primers should be provided.
Melting temperature: 55 – 58°C
Optimal length: 18 – 25mer

Primer base composition: G or C should be at the 3’ end, try to avoid more than 3 G’s or C’s at the end and 4 identical bases in a row.

Order form: be sure to fill in sample concentration. StarSEQ will not measure the DNA concentration of your samples. If sample concentration is not specified StarSEQ will take 100 ng/µl as a basis. For V-mix plate please provide sample concentration together with sample name separated with forward slash for each well.
Tube/Plate: 1.5 ml tubes (also for primers or standard 96 well plate sealed with PCR foil, aliminium foil or strips
Label: legible, with indelible black pen (e. g. Staedtler permanent lumocolor; Art. Nr. 318-9; EAN 40 07817 304563). Note your abbreviation on the lids and number them continuously. Please do not use special characters for the sample names like /[\}&%. Underscores and hyphen could be used.
Dispatch: Protect the tube with suitable firm boxes and send them in stable padded envelope.

*All quoted prices are without tax.

Sequencing results: sequencing data (.ab1 and .seq files) will be send to you by email and can also be downloaded directly from your account. The sequencing results are stored in your account for three months.

Hints for successful sequencing:

• Sanger sequencing is not a PCR! Please add only one primer, otherwise you will receive a heterogeneous sequence.
• DNA for sequencing should always be washed 2-3 times! It is generally not sufficient to wash the DNA only once.
• Spin the columns during the washing steps for 2 minutes, 30 sec is not enough.
• After the last washing step, the final spinning time should be 3-5 minutes.
• Be sure that your centrifuge reaches the acceleration needed in g (not rpm). If the centrifuge does not reach this acceleration, you need to prolong the spinning time.
• Before eluting the DNA place the column with open lid on top of the bench for 10-15 minutes to allow for evaporation of residual traces of EtOH. This step is very critical. The most common problem in Sanger sequencing is caused by EtOH contamination resulting in fast decreasing signals and short read lengths.
• Do not elute DNA in the buffer that comes with the kit, try always to elute in molecular grade water.
• Avoid overloaded columns during plasmid preparation or gel elution. Do not try to get as much DNA as possible out of the preps. This will lead to massive contamination from cell debris/gel in the eluted DNA. The output DNA may work in PCR, but the quality is too poor for sequencing.
• Check the TM of the primer; it should be 52 – 58°C. Please note that sequencing conditions are different from most PCR conditions. The final concentration of salt in the sequencing reaction is 80 mM Tris, 2mM MgCI2. Please check the TM using these conditions.