Samples must be purified after first PCR and delivered exactly in required concentration. Too low a concentration could lead to a low PCR yield, high concentration to different yields or even inhibition of the reaction (high DNA concentration may be associated with a high concentration of inhibitors). Please provide samples in 0,2 mL strips or 96-well plates.
It is required that samples are provided with tails (overhangs) as described, purified from artifacts such as excess primers or adaptors, enzymes etc.
Total lenght of DNA fragments must be between 200 and 900 bp!
How to design primers
Append to 5’ end of your primers:
Forward: 5’ TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-[locus specific sequence]
Reverse: 5’ GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-[locus specific sequence]
We use ShareSeq sequencing, where the sample processing cost is at a tolerable level.
Because our specimens are very specific (non-cultivable RNA viruses), it was necessary to create our own procedure. In this case, SEQme company worked amazingly with me, so that the first test specimen was successfully processed. In any case, we can recommend SEQme for both routine sample sequencing and more demanding projects requiring modified procedures. They are very flexible and creative.
Romana
Veterinary Research Institute, Brno, Czech Republic