Submitting DNA templates for Sanger sequencing
Number of samples:
- Fewer than 21: may be submitted in microfuge tubes. Tubes should be clearly marked with initials and sample number (eg. AB1, AB2, AB3, etc.).
- More than 21 samples: must be submitted in a 96 well plate. Wells A01 and B01 should be left empty. The plate should be loaded by columns, i.e. fill C01 through H01, then A02 through H02, A03 through H03, etc.
The templates should be dissolved in Elution Buffer lacking EDTA. Alternatively, a modified TE buffer with 0.1 mM EDTA can be used.
- Plasmid DNA: should be supplied at a concentration of 20-200 ng/µl. Typically 20 100 ng is used for each sequencing reaction.
- PCR Products: should be supplied at a concentration of 4-20 ng/µl. Typically 10 50ng is used for each sequencing reaction. PCR products must be purified before cycle sequencing to remove excess primers and enzyme. This service can be provided by UKHC Genomics Core Laboratory.
- Cosmids and Fosmids: should be supplied at a minimum concentration of 200 ng/µl. Each sequencing reaction requires 1 µg of fosmid/cosmid template.
- UKHC Genomics Center has a number of standard primers on hand (see primer list). If a template-specific primer is required, it should be provided at 5-10 µM concentration.
- Note: UKHC Genomics Center uses 3.2 pmol of primer in each sequencing reaction.
Completed sequencing reactions
- If sequencing reactions have been performed the client and are being submitted for RUN ONLY, it is best to submit the samples as dry pellets (if ethanol precipitation has been used to clean up sequencing reactions).
- If another method of cleanup has been performed, we can add Hi-Di formamide to a final concentration of 25%.
Sequencing reaction cleanup
Sequencing Reaction Cleanup Ethanol/EDTA Precipitation (10 µl reaction volume)
- Remove 96-well ABI plate from thermocycler and pulse spin at 1000 rpm.
- Add 2.5 µl of 125 mM EDTA to each well.
- Add 30ul of 100% ethanol to each well.
- Seal tightly with plate seal; mix by inverting 4X.
- Incubate 15 minutes at room temperature
- Centrifuge 4000 rpm (3000 rcf) for 15 minutes at 4°.
- Immediately* pour off EtOH supernatant with a quick inversion and gentle shake.
- Add 30 µl of 70% EtOH to each well and centrifuge at 3000 rpm (1690 rcf) for 15 minutes at 4°.
- Immediately* pour off supernatant and invert on tri-folded paper towel.
- Place in centrifuge (inverted on paper towel) and pulse spin up to 200 rpm to remove excess EtOH. (Stop the spin when speed is ~ 150 rpm, as the machine will continue to accelerate briefly after the pulse is stopped - DO NOT ALLOW SPEED TO GO HIGHER THAN 500 rpm.)
- Dry sample plates (right side up) under hood in the dark for 1 hour.
*Otherwise spin an additional 2 minutes.
If you are unable to deliver samples to UKHC Genomics Center or the Drop Box immediately, then seal and store as a dry pellet at -20°
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