Work_Order

SREL DNA Lab

Microsatellite Development Work Order

(print out, complete, copy for yourself, and send)

Samples included:

Option 1 - 4) I am enclosing two samples of high molecular weight DNA (≥150 uL of ≥50 ng/uL of >10kb DNA per sample). A picture of a ___ uL [write in amount] aliquot run on a 1% agarose minigel is enclosed. Please provide information about the ladder/size standard used. We generally recommend using a ladder for a size standard and lanes with 10, 50, and 200 ng/uL of uncut lambda to quantify the amount of DNA. Run times can be kept relatively short (~20-30 min.), as absolute sizing isn't that important.

Option 4) ≥22 additional DNA samples suitable for PCR (≥150 uL of 20 ng/uL). A picture of a ___ uL [write in amount] aliquot run on a 1% agarose minigel is enclosed.

Summary of work & limitations:

Option 0) We will send you aliquots of our reagents so you can do the enrichment yourself. Please download our latest protocol.

Option 1) We will Digest the DNA, Ligate the DNA with Linkers, Enrich with Biotinylated Probes, and Recover the Enriched DNA via PCR. We will send you: a) DNA eluted after enrichment, b) PCR amplicons from the eluted DNA (i.e., recovered DNA), c) SuperSNX-f so you can recreate (b) from (a), fresh for TA cloning, and d) copies of all gel pictures. Generally, at least 2 of libraries yield > 50% of clones with microsatellite repeats. We guarantee that at least one of the 3 libraries will have > 25% of clones with microsatellite repeats.

Option 2) In addition to option 1, we will TA Ligate the Enriched DNA, Transform the TA Ligation, Screen Plasmid Insert Size, and Sequence at least 96 Plasmid Inserts of Appropriate Size. We will ship aliquots of colonies and/or plasmids identified with microsatellites. We will guarantee that at least 64 insert sequences will form contigs and that at least 24 of the sequences have microsatellite repeats (if they don't, we'll do more sequencing and/or repeat the enrichment one time).

Option 3) In addition to option 2, we will Edit Sequences into Contigs using Sequencher, Identify microsatellite repeats with msatCommander, and Design Primers using autoPrimer. All edited contigs will be posted to a secure web page (password protected folder, not https), along with msatCommander results, and a summary of primers designed. We can not guarantee what proportion of primer pairs designed will be amplifiable in your lab, what proportion will yield scorable results from the genomic DNA of your critters, or what proportion are polymorphic, etc. But, this option is free – so what more could you really ask for?!

Option 4) In addition to option 3, we will order primers for at least 24 loci. We will attempt PCR optimization for all loci, and screen the successfully optimized loci for the presence of polymorphism among 15 individuals. We will guarantee that at least 16 primer pairs amplify a fragment of the expected size from the plasmid sequenced. We will also co-author (with you) a primer note describing these results (if at least 8 primer pairs are polymorphic). We will do our best to identify 8 polymorphic loci, but we cannot guarantee that because some critters simply don’t have “normal” levels of polymorphism.

Disclaimer: Please note that prices and our ability to do any of this work assumes that SREL will continue to remain operational. Since January 2001, the SREL budget has decreased ~60% and has been targeted for complete elimination by personnel in DOE headquarters (Washington DC) at least 3 times. SREL continues to receive strong public and scientific support for our mission, but if SREL ceases to operate or if significant personnel choose more stable employment, then any or all microsatellite development projects may cease at any stage. We will do our best to get information & DNA back to you, but we simply can’t guarantee that because the armed guards take orders from DOE and the President, not us.

Costs: (note prices are for each step and are in U.S. dollars)

Option 0) Aliquots for enrichment, plus protocol and list of other reagents needed.
a) $200 for 50uL of 3 oligo mixes; x _____ aliquots = $__________
b) $250 for 50uL of 3 oligo mixes + superSNX linker & primer; x _____ aliquots = $__________

Option 1) Enriched DNA.
a) $1750 for double enrichments of 3 oligo mixes; x _____ species = $__________

Option 2) Sequenced inserts obtained from Option 1.
                    a) $4000 for 96 colonies; x _____ species = $__________
                + b) $2000 for each additional group of 96 colonies; x _____ # of groups = $__________
                + c) $500 surcharge for subsequent orders of additional clones; x_____ # of groups = $__________

Option 3) Primers designed from sequences obtained in Option 2
[note: additional sequencing may be necessary to design the requested number of primers].
a) Free – Lucky You!

Option 4) Synthesis of 20 primer pairs obtained from Option 3a) and loci screened.
a) $4250 for > 20 loci; x _____ species = $__________
b) Contact us for prices and availability.

If it is easier, feel free to highlight or circle the appropriate choices below, and then calculate a total.

Multiple Species Microsatellite Development Pricing
Prices are in US Dollars
	1 species	2 at once	3 at once	4 at once
Double Enrichment	1750	2500	3000	3500
Sequence 1 plates of clones	4000	7000	9000	11000
AutoPrimer	free	free	free	free
Test 20 primer pairs on 23 DNAs	4250	7500	10000	12500

Total

Contact for DNA samples --
Name: _____________________________________
Shipping address: _____________________________________________________
Email: ___________________________________
Phone: ____________________________
Fax: ___________________________
Species name: ____________________________
Common name: ___________________________________

Contact for payment issues --
Name: _______________________________________
Address: _______________________________________________________________
Email: __________________________________________
Phone: ______________________________
Fax: ____________________________

Any other people associated with the project who we need to know about --
Name(s): _____________________________________________________________
Address: _________________________________________________________________
Email: _________________________________________
Phone: ___________________________________

Prior to shipping any DNA, please read the page on payment (link), and send an email to msatdevo@gmail.com. Attach photos below/on back .

Total Amount enclosed (if any; we are happy to invoice) $___________________________

Mail to (& ship internationally to):
Cris Hagen
Savannah River Ecology Laboratory
PO Drawer E
Aiken, SC 29802

Courier address (within U.S. only):
Cris Hagen
Savannah River Ecology Laboratory
Savannah River Site, Building 737-A
Aiken, SC 29808

I __________________________________________________________________________ [print name & institution]

understand and agree to the above terms and conditions, as well as those outlined on the web pages.

_______________________________________________ [sign & date]

Revised 14 September 2008
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