Your DNA template must be very clean for successful sequencing. Make sure you follow recommended methods of preparation and adjust your concentrations properly. Don’t assume that because a particular kit or protocol for isolation works for another lab procedure, it will work with our system. It may not. See our list of recommended purification methods for information on what will work best for you.
Resuspend your DNA template in very clean dH2O. Do not use TE! The EB buffer that comes with the Qiagen kits will work if it does not contain EDTA. The EDTA cheletes the magnesium ions required for our enzyme to work.
Do not use JM109 as a host strain. It makes excessive amounts of protein and it is difficult to get a preparation free of protein contamination. We recommend DH15alpha or HB101.
Protein contamination and improper concentrations are the most frequent causes of inadequate sequence. In addition, improperly prepared sample can be detrimental to our capillary array. Please make every effort to give us high quality samples!
Concentrations and Volumes
Concentrations are important! Please don’t submit samples that are not at the requested concentrations!
Plasmids: 50-75 ng/ul Please provide 4-5ul for each reaction.
Primers: 1.6uM (1.6pmol/ul) Please provide 4-5ul for each reaction.
The best primer parameters are:
Tm = 56C to 65C
%G/C = ~50%
At least 18 bases long.
PCR Products: All must be purified. Excess primers, salts, or taq interfere with the sequencing reaction.
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