How tos and FAQs

StandardSeq – Preparation of samples

Sample is a template mixed together with sequencing primer in a single test tube / well or template only if you wish to use some of our universal primers. Template is a PCR product or plasmid.

Number of samples ordered = number of tubes sent or number of plate wells used.

All templates must be purified (e.g. plasmids on cleanup columns or using other suitable method compatible with sequencing, PCR products enzymatically using Exo-SAP or otherwise), or at least precipitated with ethanol, and dissolved in water (HPLC-grade). We do not recommend buffers (TE, etc.) for dilution.

 

If you use your own primers, mix the following:

Template

Amount

Your primer

Final volume

PCR product <500 bp

50 ng

Add 25 pmol of
your primer (5 μl
of 5 μM primer)

10 μl

PCR product 500–1000 bp

100 ng

PCR product >1000 bp

200 ng

Plasmid

500 ng

 

If you wish to use our universal primers, prepare the template as follows:

Template

Amount

Final volume

PCR product <500 bp

50 ng

5 μl

PCR product 500–1000 bp

100 ng

PCR product >1000 bp

200 ng

Plasmid

500 ng

 

If you do not have the required amount of template available, you can reduce the total volume of sent samples while maintaining the concentration of template and primer. E.g.: If you have only 30 ng of PCR product of 400 bases, use 3 μl of this template and 3 μl of 5 μM primer solution. The total volume in this case will be 6 μl. Please note that in such cases we cannot guarantee the repetition of sequencing if the first sequencing reaction fails, whatever the reason.

The minimum total volume of template + primer you can send is 4 μl.

Please do not use parafilm or adhesive tape to secure your plasticware.

Download sample prep guide in pdf.

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