- High copy [plasmid] vectors. Standard alkaline lysis followed by silica/glass-fiber column purification uses to give approppriate results.
- Low-copy/large vectors. Best results are obtained with ultrapure [anionic exchange] commercial systems.
- See concentration table and volume requirements below.
- If the PCR yields unspecific fragments, the specific fragment has to be purified after fractionation by electrophoresis. It is important then: a) to limit or avoid UV exposure, and b) to recover enough material (see concentration table below).
- If the PCR gives only the specific product, no fractionation by electrophoresis is needed. However residual dNTPs and primers should be removed. Alternative methods can be used for this purpose: enzymatic treatment (Exo1+SAP), silica spin-column adsorption, ultrafiltration.
- Any of the PCR primers can be provided as a sequencing primer. If a cleaner result is needed, a sequencing oligo extending a minimum of 3-5 bases 3′ from the end of the PCR primer may save the need of further template purification. If both ends are desired, bring both primers in separate tubes (never mixed). Otherwise please follow indications below.
Template concentration (cloned insert or PCR product)
|100-200 pb||3 ng/µL|
|300-600 pb||6-12 ng/µL|
|700-1500 pb||14-30 ng/µL|
|> 1500 pb||40-100 ng/µL|
|BAC, Cosmid||500-1000 ng/µL|
These requirements refer to concentrations measured by UV spectrophotometry (i.e. Nanodrop.) Beware spectrophotometric analysis is sensitive to impurities, which can give gross 260nm overestimations for low concentrations (below 10 ng/µL). Please use the more accurate fluorometric methods instead, if available, or assessment by gel electrophoresis with a mass DNA ladder (NEB or ThermoFisher). Those target values should be halved should they be measured by fluorometry (e.g. Qubit from Invitrogen or Quantus from Promega).
- The Service provides free of charge generic vector primers such as M13Rv, M13Fw, T7 or SP6. Primer availability can be checked in the Excel table sequencing primer list.xls.
- Provide at least 10 µL of primer solution at 3 pmol/µl (3 µM) in ultrapure water or 0.1 mM EDTA pH8, in an independent tube and properly labeled. Add 2 µL (6 pmol) more per every additional template to be sequenced from the same primer.
- Oligonucleotide primers MUST NOT be labeled, fluorescently or otherwise, nor modified in any form that might interfere with the sequencing process.
- Binding site should be located at least 50 bases from the start of the sequence one whises to determine.
- Tm above 55ºC sequence-dependent value calculated with the following formula:
Tm= 66 +0,55(% G+C) -675/L
(where “% G+C” is the GC content (%), and “L” its lenght)
- Length from 17-mer onwards (until it reaches the minimum required Tm.)
- Avoid homopolymeric stretches longer than 3 bases, specially at the 3′ end.
Results are delivered in about two working days after sample reception. The user receives a notification by e-mail, including a results download link, once the service is completed.
How to download sequence results
Please follow the indications in the notification e-mail.
Sequence results remain on the server for 15 days.