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Biopolymers Facility at Harvard Medical School

The Genotyping core is available for microsatellite
genotyping of the mouse and human genomes. All work is project based and
designed in consultation with our technical staff.
If you have any questions, please contact our
Genotyping core technician at: genotype@genome.med.harvard.edu
The genotyping core is available for microsatellite marker
analysis using our 3730xL DNA Analyzer. The Biopolymers Facility maintains a
stock of labeled primers for numerous loci covering the mouse genome and human genome
at a 10cM density. Other individual markers may be purchased from ABI.
Custom markers may also be used if supplied by the client or we will design order
and validate primers for an additional fee.
All standard primers are labeled with 6-FAM, VIC or NED dyes, and are available through
Applied Biosystems.
The primers for microsatellite genotyping are designed from
DNA sequences adjacent to regions of tandem repeat units. The variable length
of these repeat regions produces polymorphic allele fragments during PCR that
can be analyzed through capillary electrophoresis. Along with size differences
we take advantage of three different fluorescent channels to run a multiplex
analysis of your fragments in a single capillary.
The BPF has, in house, the entire Mouse Marker Panel and Panels 1 - 28 of the Human Linkage Mapping Set. Any primer may be ordered individually.

A complete lists of primers are available as Adobe PDF files:
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The standard service for genotyping includes a labeling
PCR reaction, ROX size standard and analysis on the 3730xL. When analysis is
complete your data will be posted to our secure FTP server as a ".fsa"
file which can be opened with GeneMapper. For the convenience of our
clients who wish to provide their own labeled products we have divided our
charges in two parts: PCR and Analysis.
| Standard Service |
| PCR labeling: |
$1.00/sample |
| 3730xL Analysis*: |
$0.50/capillary |
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* Note: This is the maximum potential charge per capillary.
Under normal conditions we will multiplex three labeled products in a single run
using the different dye sets. Depending on size differences between the expected
fragments we may run nine markers or more per capillary.
| Marker Name |
Size Range |
Dye |
Pool# |
| Marker1 |
91-97 |
NED |
1 |
| Marker2 |
150-154 |
VIC |
1 |
| Marker3 |
200-210 |
FAM |
1 |
| Marker4 |
98-104 |
FAM |
1 |
| Marker5 |
120-132 |
NED |
1 |
| Marker6 |
190-210 |
VIC |
1 |
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Since each marker in one color is more than 20bp away from the other,
they can be multiplexed into one pool.
Cost: per DNA sample
| Each Marker |
$1.00 |
$6.00 |
| Capillary Run |
(1 pool) |
$0.50 |
|
Total: |
$6.50 |
|
Per marker average: |
$1.08 |
| Marker Name |
Size Range |
Dye |
Pool# |
| Marker1 |
100-105 |
FAM |
1 |
| Marker2 |
102-110 |
FAM |
2 |
| Marker3 |
108-120 |
NED |
1 |
| Marker4 |
106-140 |
FAM |
3 |
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Since 3 of the 4 markers are FAM in overlapping sizes,
they must each be put in a separate pool. The NED can multiplex with any of them.
Cost: per DNA sample
| Each Marker |
$1.00 |
$4.00 |
| Capillary Run |
(3 pools) |
$1.50 |
|
Total: |
$5.50 |
|
Per marker average: |
$1.37 |
- Primer will be deigned using primer3 software and ordered.
- When they arrive, they will be validated using a standard PCR reaction and then run on an agarose gel.
- If primers fail validation, they will be re-designed and re-ordered.
- This will continue until we have working primers, or until we do not feel any further design will be of any benefit.
- Once we have successful primers. we will order the fluorescently labeled primers.
- We will validate on a few of your DNA samples(8-16). Once we are convinced they will perform well, we will continue and complete the project.
| Service |
Price |
| Non-labeled primer pairs |
usually $10-20 |
| Labeled forward primer |
usually $100-150 |
| Primer design costs |
$35/hr |
| Validation costs |
$0.60/reaction |
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The final concentration of template should be 50ng/ul. For each loci you will need 1.2ul total volume. (60ng DNA)
You may select primers from our stock sets.
If you are not selecting from our stock sets, you have two options. You may design, order and provide the primers to us.
Otherwise, primers may be designed, ordered and validated by the BPF for an additional cost.
Clients who have fluorescently labeled microsatellite fragments
using Applied Biosystems chemistry may submit whole plates for 3730xL analysis.
Please re-hydrate your samples in Hi Di formamide and be sure to include a size
standard. You will need to generate a plate sheet based on our template file and
then e-mail your completed data table to our genotyping core at:
genotype@genome.med.harvard.edu.
All 3730xL fragment analysis samples should be prepared in an ABI compatible plate such as ABgene/Marsh T0296PE with a total volume of 12ul.
Please contact us for 384 well format information.
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