SREL
DNA Lab
Option 3 - Designed Primers for Microsatellites
Overview:
E-mail Glenn@srel.edu to determine when/if we can
schedule you in.
Send us:
2 tubes of DNA per
species (≥100 µL of >50ng/µL of high molecular weight DNA)
a signed work order
Here are the details:
1) Download a copy of our enrichment protocol from the SREL DNA Lab Protocols web page. You are free to
follow the protocol yourself, or we can do the enrichments for you; you will
want to have the protocol either way.
2) You will have to do step I - Isolating the DNA. You will need to
isolate ~10 ug of high molecular weight DNA (ideally more than 100 µL of
>100 ng/µL, but 50 ng/µL will work fine; and even 20 ng/µL usually works).
We recommend sending 2 or 3 good DNA samples per species of interest. You can
use Qiagen or Wizard or PureGene kits or do PCI extractions - do whatever works
best for you. Before sending the DNA to us, run out an aliquot of DNA on a 1%
agarose gel along with lambda DNA - about 90% of the DNA should be the same
size as the lambda DNA. If you
have questions, e-mail Travis (Glenn@srel.edu).
3) We will do steps II - V: Digesting the DNA, Linker Ligation, Enrichment
with Biotinylated Probes, and Recovery PCR. Please see the current list of probes
available. We do serial/double enrichments with 3 different probe mixes.
A variety of probe
mixes are available, but we will generally only modify the mixes used
for large-scale projects (i.e., projects needing several hundred primer pairs).
4) We will sequence inserts from a plate of clone. We generally have
>80% of clones sequence successfully on at least one strand. We will import the sequences into
Sequencher, trim off the vector, and trim off poor quality bases. We will then form contigs by clone, and
then try to contig the clones (to find duplicates). This will be done using automated protocols & should
be checked for accuracy! We will then export the sequence to a
concatenated fasta file which will be screened for the presence of
microsatellites using msatCommander. If you request it at the time of ordering, we will ship the
items mentioned under option 2.
5) From the sequences containing microsatellites identified from the 96
clones sequenced (see option 2), we will design
primers for all possible primer pairs using AutoPrimer. This program
is courtesy of the genius and generosity of Brant Faircloth at UGA.
6) We cannot guarantee what proportion of
primer pairs designed will be amplifiable in your lab, what proportion yield
scorable results from the genomic DNA of your critters, what proportion are
polymorphic, etc., but we’re doing this third option for free. So, really, what more could you
realistically expect?
7) Please refer to the Order Form for
current prices. These are considerably less than commercial sources. As part of
this "bargain" you may have to tolerate potential delays on our part
(we will strive to obtain results in 12 weeks, but WE
DO NOT GUARANTEE that schedule). Contemplate realistic
expectations for “free services” & we promise to exceed them!
Website content by Travis Glenn
For more information, contact: Glenn@srel.edu
Back to Microsatellites - home.
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Option 3 - |
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Launched 14 December 2001
Revised 2 April 2007