UH Hilo Core Genetics Facility: DNA Sequencing Service
Sequencing Sample Prep
1. DNA Template Preparation: Use appropriate purification protocols; prepare sufficient template
to allow for its accurate quantitation and purity verification
a. For PCR products: use QIAGEN QIAquick PCR purification protocol, Exo Sap, columns
(or equivalent)
b. For plasmids: useQIAGEN QIAprep plasmid purification protocol (or equivalent)
2. DNA Template amount: the amount of template depends on the form of the DNA
a. dsDNA plasmid:
50-600 fmol
purified PCR product:
(see sequencing request form for qty)
b. primer concentration: 3.5 – 5.0 pMol or 1 µL of a 3.5 – 5.0 μM primer in a 10 µL
reaction.
i. include only one primer for sequencing (i.e. for sequencing in forward OR reverse
direction), if both primers are added, sequencing reaction will not work!
3. Template Pre-Heat Treatment: For certain plasmid DNA templates, the following pre-heat
treatment improves signal strength and current stability
a. Dilute template with water to the appropriate concentration (do not add primer or other
components until after pre heat treatment)
b. Heat the template at 96˚C for 1 min, and cool to room temp before adding the remainder of
the sequencing-reaction components
c. alternative pre heat treatments: heat at 86˚C for 5 min, or 96˚C for 3min
4. Final Sample to be submitted: prepare in sequencing plate
Component
for 1/4 reaction
DNA template
see request form
Primer (3.5–5.0 pMol)
x μl
dH2O
x μl
Total
7.5 μl
5. Along with the sample to be submitted, please fill submit DNA Sequencing Sample Sheet, and
indicate how data is to be returned (email or USB drive), and submit appropriate data storage
device.
6. Billing: Payment is not due until the end of each month or upon agreement, at which time total
services provided by the Core Facility will be added. An invoice will be generated and sent to the
address indicated on the DNA Sequencing Request Form.
Core Facility Services Procedure:
DNA sequencing reaction:
1. After sequencing samples are submitted, the AB BigDye Terminator v3.1 Cycle Sequencing
reagents are added to the samples (2.5 ul for a ¼ reaction, for a total volume of 10 ul).
2. Sequencing reaction thermal cycling program:
96˚C for
1 min
96˚C for
10 sec
50˚C for
5 sec
60˚C for
4 min
For 30 cycles, followed by holding at 10˚C
Sequencing reaction clean-up and Sample prep:
1. After sequencing reaction is complete, sample needs to be purified in order to remove unused
primer, dNTPs and ddNTPs
2. BigDye XTerminatorTM Purification Kit is added directly to the samples, and plate is placed on a
shaker for 30 minutes after which the plate is added to the sequencer.
AB 3500 run and Sample Results:
1. Sample plate is loaded into the AB 3500 Genetic Analyzer
2. Sample sheet (containing sample IDs) is prepared with the appropriate run method assigned
3. Sequence run is initiated; Method is run and raw data is collected (current and voltage readings,
and a log of activity are also recorded)
4. Raw data is normalized and automatically converted to the final “Sequence Results”, and
presented as a chromatograph.