“There’s never time to do it right but there’s always time to do it over.”
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.
Do not use TE! Resuspend your DNA template in very clean dH2O. 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.
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!
Qiagen publishes a very good template purification guide. It would serve you well to download and read it. Please ignore its advice on genomic preparation as we do not accept genomic preps.
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.
PCR Products: Please see our Sequencing PCR Products Page for concentrations and volumes.
Recommended Methods of Purification
The mere use of the kits we recommend does not guarantee success. You must still use good technique!
- Qiagen Spin Kits- (Available in our Qiagen Cabinet)
Note: Always do the optional wash even if your strain is not part of the JM series. The elution buffer that comes with the kit works well for sequencing. No need to use dH2O.
Qiagen publishes a very good template purification guide. Please ignore its advice on genomic preparation as we do not accept genomic preps. If you are having trouble getting yield with the Spin kits: Elute with hot dH2O (60C-70C) that has been recently autoclaved.
- Promega Wizard SV
- Bio-Rad Plasmid Miniprep Kit
- Nucleobond AX Cartridge
- Novagen UltraMobius 200 Plasmid Kit: Our thanks to John Beard in Timothy O’Brien’s lab for testing it! It works great!
- Mini-alkaline lysis + PEG precipitation Note: This is labor-intensive, requires some skill, and nobody uses it anymore. You’re better off purchasing a kit.
Purification Methods Not Recommended
- Any Qiagen MIDI or MAXI kit! Some people can make these work for sequencing but we do NOT recommend them. They have proven to be unreliable and inconsistent for sequencing.
- S & S DNA Elution Beads
- Stratagene Strataresin
- Isoquick Plasmid Kit
- Classical Alkaline Lysis
- 3′-5′ Plasmid Isolation Kit. Although Perkin Elmer recommends it, it seems to be very inconsistent.
- Gibco’s Concert Prep.
- BIO 101 Genekleen
- CsCl purification