A. MGIF Stock Primers
IBL Stock Primers list
B. Custom Primers
When designing a custom primer for sequencing, there are a few considerations to make before having the primer synthesized. First, ideally design a primer that is between 18 and 24 bases long. Second, check for secondary structure of the primer. This can be done with a number of different programs (ie. Oligo and Gene Runner, etc.). The primer should be free of all types of secondary structure (hairpin loops and dimer formations). However, this is not always possible.
Based on our observations you need to follow some basic rules. First, make sure that all hairpin loops are at least six or more bases away from the 3' end. Make sure that any dimer formations present are at the 5' end and not the 3' end. Finally, the annealing temperature (Tm) of the primer should be between 45 and 55 degrees Celcius. If the Tm falls outside of this range, then you need to let us know the sequence of the primer so that we can verify the Tm and run an alternate cycle appropriate for your reaction.
C. Primer Walking a Template
When designing a primer from a template, make sure that you DO NOT select a primer in an area where you cannot positively identify the base. The best region for designing a primer from a sequence varies from reaction to reaction. No matter what region you use to design your primer, make sure that you use the criteria listed above for custom primers when it comes to length, secondary structure, and annealing temperature.
Also make sure that the primer is at least 50 bases away from the area that you are interested in sequencing. Remember that each mismatch between the primer and template decreases the quality of the sequence if you obtain a sequence at all.
D. Purity
Typically, the best sequences use purified primers. However, this does not mean that an unpurified primer will not work, but your chances for failure are greater.
The easiest way to purify a primer is to have it purified during synthesis. After synthesis, your options for purification are reduced to either HPLC or gel purification.
E. Age
Primers that are stored in solution over six months will probably degrade. As a result, you will want to use a primer that is less than six months old for the best results. Our standard primers are changed out monthly.
F. Concentration
Primers should be submitted to us in HPLC grade water with the concentration dependant on the secondary structure of your primer. If you are not sure or you know that the primer has some form of secondary structure, send us 25 uL of a 30 uM solution (30 pm/uL). Otherwise, send us 25 uL of a 4 uM solution (4pm/uL) if the primer is free of all types of secondary structure.
We do not check the concentration of your primer. We are depending on you to send the appropriate concentration. Please do not send a solution that is greater than 50 uM. Some sequencing results suggest that a concentration over 40 uM may interfere with the sequencing reaction.