DNA Sequencing - Custom Sequencing Service
We can perform all the necessary cycle sequencing procedures as well as the electrophoretic separation as part of this service. You must supply sufficient quantities of the DNA Template and primer for each reaction.
Template
As stated in our chemistry section, template purity is vital for achieving the best possible result. Additionally, contaminated template DNA can permanently affect the linings of our sequencer capillaries resulting in a loss of basecall resolution.
We require that ALL template DNA to be used in the cycle sequencing reactions submitted to our facility, be post-extraction purified to eliminate the contaminants that cause capillary degradation and reaction failure.
We prefer that you use one of the many standard, commercially available column-based, DNA purification kits that are appropriate for the type of template you are using. We can provide a list of commonly-used products and suppliers if needed. The minimum standard acceptable at Micromon is the ABI alkaline lysis-PEG precipitation method.
We require the following amounts of template per cycle sequencing reaction:
| 5µl @ 100ng/µl |
Single-stranded templates |
| 5µl @ 200ng/µl |
Double-stranded templates |
| 5µl @ 10ng/µl |
PCR products < 1 kb |
| 5µl @ 20ng/µl |
PCR products > 1 kb |
| 10µl @ 20ng/µl |
PCR products > 2 kb |
Note: It is critical that the estimate of DNA concentration is as accurate as possible. Due to the presence of contaminants in mini-preps of plasmid DNA, even with column purification, we strongly recommend that you estimate your template DNA concentration by gel electrophoresis determination.
Primer
We require 5ul at 3-5 µM (3-5 pmol/µl) for each reaction. In addition to high template purity, good primer design is a significant factor in successful DNA sequencing.
Our facility can supply the following common primers at no extra cost. Please check that the sequence of our in-house primers matches the priming sequence in your particular cloning vector.
| M13/pUC For Primer (-21) |
5' TGT AAA ACG ACG GCC AGT 3' universal |
| M13/pUC Rev Primer |
5' TCA CAC AGG AAA CAG CTA TGA C 3' |
| T7 Primer |
5' TAA TAC GAC TCA CTA TAG GG 3' promoter |
| T7 Rev Primer |
5' GCT AGT TAT TGC TCA GCG G 3' terminator |
| SP6 Primer |
5' TAT TTA GGT GAC ACT ATA G 3' promoter |
| T3 Primer |
5' ATT AAC CCT CAC TAA AGG GA 3' promoter |
Note: Despatching Orders to Micromon
Please send your template DNA and primer in separate 1.5 ml microfuge tubes, together with a completed order form. We recommend that you ensure that either the tubes are sealed with Parafilm or that you use screw cap tubes to minimise evaporation or accidental loss of sample during transit. Orders can be delivered in person, by courier or by post.
We offer a combined arrangement where your custom primers can be synthesised on site, prepared and used directly in the cycle sequencing of your template DNA. This will save you valuable time and money. You only need send your combined order to the Oligonucleotide Synthesis Facility together with your template(s).
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