All microsatellite loci are perfect repeats (n > 12 for di-nucleotide repeats and n > 6 for tetra-).
All of the primers in DOGSET are designed using standardized parameters:
|Product Size:||100-400 bp|
|Primer Size (min)||19|
|Primer Size (opt)||20|
|Primer Size (max)||22|
M13 labeled sequences: FAM = TTT CCC AGT CAC GAC GTT G NED = TAA AAC GAC GGC CAG TGC VIC = GCG GAT AAC AAT TTC ACA CAG G
Identical sequences appended to the 5' end of the locus-specific forward primers. For a description of the M13 tailed primer protocol please see: M13 Tailed Primer Protocol.
The following sequence can be appended to the 5' end of the reverse primer:
+A/-A Tail = GTTTCTT
This sequence serves to drive the non-templated addition by Taq of a base (usually +A) onto the 3' end of the labeled strand. This allows for easier allele scoring (by eliminating the -A peak).
Forward/ M13 /Reverse/ are in final ratio of 0.1x/0.2x/1.0x
X = 1.8 uM
DNA - 2 µls of cheek swab extract (standard extraction protocol) added to each well and allowed to desiccate (stable for at least several months at room temperature).
Alternatively, 50 ng pure DNA (extracted from tissue or blood) is used.
Primer mix (5 µls) is added to each well and covered with Chill-Out® (Bio-Rad); allows for easy hot-start setup.
PCRs for genotyping performed in 17-µl reactions
|10x buffer||178.5 µls|
|dNTPs (2.0 mM stock)||178.5 µls|
|MgCl2 (25 mM stock)||178.5 µls|
Hot-start: DNA with primer mix (5 µls) are brought to 93°C (covered with Chill-Out®). 12 µls of cocktail mix is then added to each well for final reaction volume of 17 µls.