SREL
DNA Lab
Option 2 - Sequencing 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 無 of >50ng/無 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 無 of
>100 ng/無, but 50 ng/無 will work fine; and even 20 ng/無 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. We will then:
A) Post all unedited ABI chromatograms to a secure web page (password
protected folder, not https), along with msatCommander results,
B) Ship aliquots of colonies as glycerol stocks (on dry ice) or on
CloneSaver cards (at ambient temperatures),
C) Ship the things we send to people who choose option 1:
a) excess DNA eluted after
enrichment,
b) excess PCR amplicons from the eluted DNA (i.e., recovered DNA),
c) excess ligation (in lieu of SNX-f), and
d) copies of all gel pictures.
5) We will guarantee that you get at
least 24 insert sequences with microsatellites (if we don't find them in
our search of the original 96 sequences, we'll do more sequencing and/or repeat
the enrichment). However, we cannot guarantee the independence of these,
what proportion will be satisfactory for designing primers, what proportion of
primers work from these, etc.
6) 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 6-8 weeks, but WE
DO NOT GUARANTEE that schedule). 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 2 - |
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Launched 14 December 2001
Revised 2 April 2007