Nothing to see - just failure?

Perhaps the most difficult result to trouble shoot is the completely failed reaction. Many times customers call and ask why they don't have any data to analyse!

The reasons for a failed reaction are as many and as varied as there are failed reactions. Usually though there will be a pointers to the reason(s) for failure on the gel image. Take a close look at your failed *.scf-file. What do you see? Nothing? Really nothing? Sometime you need to expand the image! If you really do see nothing in the lane then you may well have lost the pellet - repeat the reaction with extreme care. If there are some faint signals you didn't lose the DNA at any point during the preparation. So, it's probably one of the other reasons below for the lack of data. Dirty templates perhaps are the biggest cause of failed reactions in our facility. If your DNA is not clean enough (i.e. get rid of all salt, protein, carbohydrate, and resins from kits) then your reaction will fail. At this point we might advise a number of things - if you're using our service then send your DNA to be prepped on our Robot. Otherwise try a manufacturers clean up kit taking special care at each step. Run your DNA out on an agarose gel judging its amount against a known concentration of a Lambda ladder or any other marker run on the same gel. Another contributory factor at the point is host strain choice - we advise DH5Alpha or XL1Blue. Avoid JM101. Primer problems : the cycling conditions in our protocols provide optimum conditions for the TaqFS+ to work during sequencing. This includes an annealing temperature of 50°C and sometimes primers you designed might not work well enough at this temperature. Please make sure that your primer is designed correctly, is at the correct concentration, and the primer binding site wasn't lost for some reason.