SREL
DNA Lab
Option 4 - Screened 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)
≥22 additional DNA samples suitable for PCR (≥100 µL of 20
ng/µL)
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.
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 will order primers for at least 24 loci using 5'
sequence-tailed primers. We will attempt PCR optimization for all loci
using 2 touchdown PCR protocols, a HotStart Taq, and DNA from 7
individuals. We will then screen
the successfully optimized loci for the presence of polymorphism among 15
additional individuals.
7) We will then work with you to write up a primer note. We will take the lead on writing it up, but you
will need to provide a paragraph or two about the critter. Our expectation is
that you have something in mind beyond this primer note & that you may want
some time to work on that project. We will target submission of the primer note
within one year of completing the data set.
8) We will guarantee that at least 16 of
primer pairs amplify a fragment of the expected size from the plasmid sequenced. However, we
cannot guarantee what proportion will yield scorable results from the genomic
DNA of your critters, what proportion are polymorphic, etc. To my
knowledge, no one will guarantee this - this is why they call it
"research".
9) 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 3-4 months, and we often achieve that goal, but WE
DO NOT GUARANTEE that schedule, and it isn’t
unusual for it to take 6 months or more. If you enquire about
potential schedules prior to sending your DNA, we will do our best to stick to
the target schedule agreed upon. Gentle
e-mails requesting updates every few weeks are welcome and often help move
things along.
Website content by Travis Glenn
For more information, contact: Glenn@srel.edu
Back to Microsatellites - home.
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Option 4 - |
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Launched 14 December 2001
Revised 2 April 2007