Use the following concentration and volume for your samples:
|
Sample type
|
Product length
|
Sample conc.
|
Vol. 1-8 rct.
|
| Plasmid DNA |
< 30 kbp |
50-100 ng/µl |
20 µl |
| BAC/PAC/Cosmid DNA |
30 - 200 kbp |
200-1000 ng/µl |
20 µl |
| Purified PCR Products |
150-300 bp |
1 ng/µl |
20 µl |
| |
300-1000 bp |
5 ng/µl |
20 µl |
| |
1000-3000 bp |
10 ng/µl |
20 µl |
| Unpurified PCR Products |
150-300 bp |
4 ng/µl |
20 µl |
| |
300-1000 bp |
10 ng/µl |
20 µl |
| |
1000-3000 bp |
20 ng/µl |
20 µl |
Premixed samples (a mixture of template and primer):
- Templates should consist of 15 µl purified DNA with either of the concentrations given in the table above
- Add 2 µl of primer with a concentration of 10 pmol/µl (10 µM)
- Ensure that the total volume of your premixed sample is 17 µl
Sending samples according to the requirements below helps us to do our job better and provides you with accurate results.
- Use 1.5 ml safe-lock tubes for your templates and primers
- Do not tape or wrap tubes with parafilm. Safe-lock tubes offer perfect sealing and evaporation protection
- Label your template with our barcode labels. Either with the prepaid TubeSeq Labels or free Tube Labels
- Label your enclosed primers with our free Tube Labels
- Use a water resistant marker for any additional labeling of your tubes
|
1
Only use 1.5 ml
safe-lock tubes
|
2
Remove barcode
stricker from film
|
3
Affix barcode sticker
horizontally
|
4
QR-code visible at front so
it can be read by scaner
|
Benefit from the greatest flexibility to handle your sequencing primers.
Thanks to our DNA synthesis lab, bioinformatic know how and internal IT solutions you can choose how to provide us with your sequencing primers:
- Select from standard primers on stock
Select and manage your favourite standard primers from more than 80 different validated standard primers
- Order specific sequencing primers
Specific primers can be ordered directly with your sequencing order. These primers are stored for 4 weeks and can be re-used during this time
- Enclose your own sequencing primers
You can send us your sequencing primers in 1.5ml tubes along with your samples by using our Primer Barcodes *
- Premix primer with DNA template
Sequencing primers can be added directly to your DNA template and sent to us as premixed sample.
Please consider optimal primer conditions, concentration and volume as described below.
Optimum primer conditions:
- Primers must not contain phosphorylation or fluorescent dyes
- The optimum primer length is between 16-25 bases
- The primer melting temperature (Tm) should be 50 - 62°C
- The GC content of the primer should be 35-60%
- Ideally one G or C should be located at the 3' primer end
- The number of 3' Gs or Cs should not exceed 2 Gs or Cs
- If possible, avoid >3 identical bases in a row in the sequence
Primer concentration and volume:
- Exactly 10 pmol/µl primer concentration is required per sequencing reaction
- Each primer must have a total volume of 20 µl (double distilled water or 5mM Tris-HCl); 2 µl of primer volume is required for every additional sequencing reaction
- Concentration of primers with wobble bases must be calculated according to the following formula: nX x ConcPrimer
DNA and primer storage:
- Storage of template DNA for 4 weeks
- Storage of synthesised primers for 4 weeks free of charge
- Storage of synthesised primers for one year at additional cost
Data storage:
- Secure online archive of all selected sequence data for 6 months
DNA Sequencing has never been so easy
Just stick one TubeSeq Label on your sample tube and enter the barcode details to identify your sample. Conveniently use our TubeSeq Coupons to pay for addtional reactions. You can also benefit from a variety of convenient online features to manage your TubeSeq Labels and TubeSeq Coupons:
Multiple options to define the rights of use
- TubeSeq Labels can be used by anyone with the same customer number
- The usage rights for TubeSeq Labels can be restricted to your account in your sequencing preferences.
- You can also define a group of people who are allowed to use your TubeSeq Labels and your TubeSeq Coupons under "My Groups".
- TubeSeq Labels and TubeSeq Coupons can be transferred to any other account under "My Sanger Kits & Barcodes"
Automatic reminder to order again
- You always see the actual number of remaining prepaid reactions under "My Sanger Kits & Barcodes" where a traffic light signals when they are running low.
- In your sequencing preferences you can select to receive a reminder by email where you can specify the critical minimum amount of your TubeSeq Labels.
Overview of status and usage details
- Under the "Prepaid Tube Barcodes" tab on the "My Sanger Kits & Barcodes" page you can find full details of your purchased TubeSeq Labels and Coupons.
- You can search, filter and sort to find specific barcodes.
- The details of users and sequencing orders are displayed in a tooltip by positioning your mouse cursor over the row of interest.
When to choose the Power Read Upgrade
In case your samples are >30kbp and/or your samples contain difficult-to-read stretches, select the Power Read Upgrade.
We will perform special cycling conditions or chemistry to achieve best sequencing results. A free repetition is included. This service requires a longer processing time.
Definition of technical failure
* A technical failure is defined as a chemical or technical (IT, machines) defect in our lab. In order to ensure that the sequencing quality is not affected by a technical failure, an internal quality control is performed with each sequencing run. In addition to that, a sophisticated software is analysing each plate considering parameters like quality values (QV) and reading lengths.
If you see "DONE_QV_NOT_MET" as a result of your sequencing reaction, it means that the internal quality control has passed, but some sample quality values have not been met. (e.g. sample or primer concentration or volume, sequence design or structure etc.).