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Template Preparation, Quality, and Quantity
Requirements
Important: All samples must be labeled with the
sample name, date, and user account number. All samples must be accompanied
by a sequencing request form indicating the appropriate service
package.
 Full-service
DNA sequencing begins with the submission of isolated and purified template
DNA and primers. Acceptable templates include double and single stranded
DNA, PCR products, and genomic DNA.
Various kits are commercially available to provide
investigators with the proper quantity and quality of template for genetic
analysis. Investigators should consider the following when choosing an
isolation method:
- Starting Material (cells, tissue, DNA fragments)
- Throughput (single tube processing or plate format)
- Expected Yield (mini, midi, maxi preparations)
- Subsequent Purification
Quantity
Guidelines for submission as per Applied Biosystems’ BigDye Terminator Cycle Sequencing protocols:
| PCR Product: |
|
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100-200 bp |
1-3ng |
5ng/µL |
200-500 bp |
v3-10ng |
5ng/µL |
500-1000 bp |
5-20ng |
5ng/µL |
1000-2000 bp |
10-40ng |
5ng/µL |
>2000 bp |
20-50ng |
5ng/µL |
| Single-Stranded |
25-50ng |
5ng/µL |
| Double-Stranded |
300ng |
100-200ng/µL |
| Cosmid, BAC |
0.5-1.0 µg |
500ng/µL |
| Bacterial genomic DNA |
2-3µg |
1µg/µL |
| genomic DNA |
50ng |
25ng/µL |
| *Customer Primers |
3-4pmol |
1pmol/µL = 1µM |
The concentrations listed are necessary for speedy processing
and investigators should plan on providing at least 2X the volume requirements
for each sequencing reaction. If your laboratory cannot achieve these
concentrations, vacuum dry concentrating is available at the Genomics
Core Facility as a custom service. Supplying extra template will ensure
that sufficient material is on hand if it is necessary to re-run a reaction.
Investigator must provide documentation confirming the concentration of
submitted samples.
* The following common vector primers are kept in stock and available from the Genomics Core Facility: -21M13Forward, M13Reverse, T3, T7promoter, BGH Reverse and Bluescript Primers: KS & SK.
** For best results genomic DNA should be subjected
to preliminary PCR reactions and purification before cycle sequencing.
Please contact
us to inquire about this service.
Quality
For optimum results, good quality template is essential. For full-service
sequencing, the investigator is expected to provide template samples with
A260/A280 of 1.8-2.0. We can include a template
quality check as a custom service prior to beginning thermal cycling.
If quality is insufficient, the investigator will be notified.
For basic service
sequencing, in which samples are provided as purified, lyophilized cycle
sequencing products, it is strongly recommended that you perform a quality
check at your laboratory, prior to sample submission, to avoid unnecessary
delays.
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