Frequently Asked Questions - Sanger Sequencing

The Premium Run is Microsynth‘s superior sequencing service in 1.5 ml tubes offering a broad spectrum of additional services such as the optimization of reaction conditions and the access to special treatment and troubleshooting protocols (e.g. alternative design of sequencing primers, one-drop sequencing in case of low DNA amounts, additional sample purification).

Generally speaking, the Premium Run is designed for customers requiring the best possible outcome along with superior service.

• Economy Run samples: 4 working days
• Premium Run samples: 3 months
• Economy Run plates: 1 month
• Your specific sequencing primers will be kept at our sequencing lab for at least 4 months (or for 10 months in case you have added them to your “Custom Primer List”).

On request, Microsynth will ship your specific sequencing primer(s) back to your or any other desired address.

Click here to open our standard primer list with all the primers you can select from.
Please note: Microsynth’s webshop allows you to maintain a customized primer list. In case you frequently use more specific sequencing primers, just send them to Microsynth and update your customized primer list. Since Microsynth operates a well-recognized oligo production facility, you can also outsource design and/or synthesis of your sequencing primers.

Yes, it is possible to send samples in one 96-well plate and request multiple reactions (with two or more primers) for each sample.

First option: identical sample from same well position with more than one primer. You send 12 ul of the sample for the first reaction plus 3 ul of the sample for each additional reaction.

You specify the sequencing reactions of the plate you're going to send physically (= master plate) indicating the first primer(s). Under "Review order" you can copy the sample pattern of the master plate (use the button "copy"). Just be aware that for our prepaid Economy Run you need another prepaid barcode label. Whereas the label number is entered into the system, the label should be attached to the master plate. Theoretically, it is possible to repeat this step several times (for further primers).

Attention:
If the master plate is not entirely filled, we recommend switching to our non-prepaid Economy Run and following the same ordering scheme (exception: use the same plate name).

Second option: identical sample from different well positions with more than one primer

If you're using the Economy Run and you want us to sequence half of the samples with the forward primer and the other half of the samples with the reverse primer you should provide the samples in two separate wells e.g. A01-H06 with forward primer and A06- H12 with reverse primer.

Up to 12 different sequencing primers per plate are added free of charge. For each additional primer, a small surcharge will be applied.

In general yes, but please note that your templates are stored for 4 working days only and overnight sequencing is not applicable in this case.
The Procedure for additional sequencing is as follows:

- Submit a new online sequencing order using the next prepaid label number
- Use the same sample name as in your previous order
- Write the following into the comment field: samples stored at Microsynth, previous order number was xxxxxx.

Remark: Once your order is processed, the used prepaid label is invalid and can be disposed of.

Dependent on the selected service type, your samples have to fulfill different requirements. For following services, Microsynth provides user guides containing a lot of helpful information:

• Economy Run
• Ecoli NightSeq
• Premium Run

Please click on the name of the service above to open the relevant user guide; or go to the “Sample Requirements” area where you will also find useful information about sample requirements etc.

• Enter our webshop
• Click on Option&Preferences in the DNA Sequencing area
• Click on Order History - Review/Delete Previous Orders

In general, failed sequencing reactions will be automatically repeated in the following mode: in a first attempt one sample will be repeated in order to evaluate the potential for further improvement by modifying the chemistry of the sequencing reaction. If the repetition yields in a better sequencing result, other samples of your order will be resequenced, too. In addition, it is always possible to ask for specific repetitions of failed reactions (best done by phone or email). Be aware, that we keep Economy Run samples only for 4 working days.

Sample- and primer names of up to 35 characters are accepted in the online order forms. Consequently, the file names of Sanger results have following format: sample name_primer name. ab1
Results from plate sequencing contain in addition the plate name: sample name_primer name_plate name.ab1
Please consider that special signs are invalid (details are listed in the web shop).

Go to the “Sequencing Primers” area where you will find a lot of useful information about sequencing primers.

Microsynth provides the sequencing results in text format (FASTA files) and in chromatogram format (ab1 files). In order to open the ab1 files you need to download one of the software tools which are provided in the Software and Links area.

By default, Microsynth is performing sequence trimming and removes misleading data from the ends of sequencing fragments. However, for certain applications (e.g. CRISPR/Cas sequencing) it may be more appropriate to obtain untrimmed sequencing data (“raw data”). In our web shop you can select under “Options & Preferences” whether you want sequence trimming or not.

Direct sequencing via the Sanger approach is possible for genomes < 1Mb. However, whenever feasible, we recommend you to amplify the region of interest by PCR and to send us the PCR product(s) for sequencing.

In order to obtain reliable sequencing results, impurities such as dNTPs, PCR primers etc. must be eliminated. Furthermore, it is important to quantify the purified PCR product. This is easily accomplished by performing agarose gel electrophoresis of your PCR product and comparing the band for your PCR product with standard bands of defined concentrations. We do not recommend quantification approaches based on UV spectrophotometric analysis techniques. For successful sequencing, PCR products must be single-banded on the agarose gel. If this is not the case, you should recover the correct band by gel extraction. Using the Premium Run we can do this for you.

The results of the Sanger sequencing are kept for at least 3 months on the webshop.