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. Mix the necessary amounts of DNA with primer, fill up to a total volume of 7 µl (if needed with water) (Please note the hints “… you will need” below!)
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 complete personal details, date of received results and the abbreviation of your samples under the following E-mail address: u-mix@starseq.com

… you will need:

DNA: dissolved in water or TrisHCl (10mM), pH 7-8 (NO EDTA!!!)

Amounts:
PCR product: < 200 bp: 50 ng; 200 – 300 bp: 60 ng; 301 – 400 bp: 80 ng; 401 – 500 bp: 100 ng; 501 – 600 bp: 120 ng; 601 – 700 bp: 140 ng; 701 – 800 bp: 160 ng; 801 – 900 bp: 180 ng; 901 – 1 kb: 200 ng
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
Cosmid DNA, PACs, BACs: >1 µg

We recommend to measure the DNA concentration with fluorescent assay or on a agarose gel.

Primer: 10 pmol, i.e. 1 µl of a 10 µMolar primer solution
Melting temperature: 52 – 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.

Tube/Plate: 200 µl PCR tubes with flat lids (e. g. Starlab, Art. Nr. I 1402-8100 or similar); Close only, no Parafilm. Always fill plates from A1 to H1, A2 to H2, A3 to H3… Samples in Plate: Its your choice (must be between 48 and 96 samples)!
Label: legible, with indelible black pen (e. g. Staedtler permanent lumocolor; Art. Nr. 318-9; EAN 40 07817 304563 or similar). Note your abbreviation on the lids and number them continuously. Please do not use special characters for the sample names like / [ \ } & % , ; : . ä ö ü ? ß ( ) ” ‘ # + * @ µ and blanks. Underscores and hyphen could be used. For U-mix please stick one barcode label for each sample to sequence somewhere on the front or back site of the printed order form.

Dispatch: Protect the tube with suitable firm boxes or bubble wrap and send them in stable tearproof padded envelope.

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.

*All quoted prices are without tax.