We have more than 20 years of experience with classical Sanger sequencing and process more than 25,000 samples per year. This allows us to keep our prices low, but you can still expect a personalised approach - we address sample preparation errors when necessary and optimise reactions for different sample types, including samples with problematic sequences.
For sequencing PCR products and plasmids, we use a 24-capillary 3500xL genetic analyzer (Life Technologies). Results are sent via e-mail in *.ab1 (chromatogram) and *.seq format within one to two business days.
Samples should be placed in the collection fridge in Viničná 7 in a box marked Biocev SEKVENACE or in the collection box at the Biocev reception.
Complete the Sample sheet for Sequencing Reaction and Analysis or the Sample sheet for Analysis only and send it to seqlab@natur.cuni.cz. Paper sample sheet do not need to be sent with the samples. The sample sheet is partially self-filling - if not, download it again.
Name the file, the subject of the E-mail, and the sample bag the same with the abbreviation and date in the form JJPDDMMYY. For example, 6.9.2024 - Ford Harrison, would be labeled HAF060924.
Label individual tubes with the abbreviation and serial number from the guide. Number logically.
Please note: 1 sample = 1 tube/well = 1 line in the guide = 1 analysis.
If you are sequencing for the first time, start with a small number of samples. Also start new series with smaller numbers of samples (e.g. after a longer time or with new primers).
If you have additional requirements, e.g., you are in a hurry for results, want results to a different address, require special conditions (higher/lower seeding temperature, Hairpin protocol, problems with previous sequencing of a similar sample, use of BigDye® Terminator v1.1, etc.), please write them in the Special requests section of the Sample sheet as well as in the email.
Instructions for sample preparation can be found here.
Purification of PCR products and plasmids prior to sequencing reactions is not performed as standard (by appointment only and for small numbers of samples, e.g. in case of doubts about the quality of purification). Repeat analyses on the genetic analyzer (if electrophoresis fails) are performed free of charge. The sequencing reaction itself cannot be repeated, the samples supplied are sufficient for one sequencing reaction and new samples must be sent in case of failure of the sequencing reaction. Sequencing reactions can fail for the following reasons: insufficient DNA purity, insufficient sample or primer concentration, incorrect primer, or, more rarely, secondary DNA structure.
When the sequencing fails, something is wrong! Do not send the same samples prepared in the same way, it will not be better! Check the purity of the DNA on the nanodrop (mainly for PCR fragments). Check that you have added the correct primer (for plasmids). Read our recommendations here. Consult with us about the situation and send DNA and primers separately. We will measure the concentration and purity of the DNA and, if necessary, purify the DNA and mix the sequencing reaction ourselves. If hairpins are expected to be present, we will recommend sequencing from a second strand, or sequencing using the Hairpin protocol, which you should indicate in the special request.
For the sequencing reaction, we currently use the BrilliantDye® Terminator v3.1 Cycle Sequencing Kit. We add Enhancing Buffer (BDX64, MCLAB) and Sequencing Buffer (Applied Biosystems) to the kit. The standard reaction runs for 35 cycles: 10 s /96 °C, 5 s /50 °C, 2.5 min /60 °C. We modify the reaction profile when the Tm primer is greater than 55°C and less than 48°C.
Samples are purified by ethanol precipitation with sodium acetate (3M) and dissolved in denaturing Hi-Di formamide for analysis. The analysis is performed on 50 cm capillaries on POP-7 polymer (Applied Biosystems). Optimally, 800 bases are obtained. Capillary electrophoresis is performed in a buffered EDTA environment.