Stock No: 002014
Protocol 14858: Pyrosequencing Assay - Ptprc<a><b>
Version 2.0

Notes

This genotyping assay uses pyrosequencing technology and is run on the Biotage PSQ 96MA. The Jackson Laboratory is not posting the complete details of our pyrosequencing genotyping assays as the primers for pyrosequencing cannot be used for sequencing using more traditional methods. The wild type and mutant nucleotides and the flanking DNA sequence are provided below.
The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX). To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility. Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.

Expected Results

Amplicon = 237 bp
HET = C/T
HOM (Ptprc<a>) = C/C
WT (Ptprc<b>) = T/T

Sequence

TCAAACTATTATCTTAAGCAAGTAGCTGTGGATATAAACTATGTCTCATTAATTTTATTTTAATACTTACCACAGCAATACCTCAAGATACCAAAACACATGGCCAACTGTGGGAATATTCTGATAGAAGAGAATATTATTGGGTAGTCTTCAGATCAGTGGGATAACTAGCCAAAAAGAATCTATGAAAGAAATCTAATCTAAATGATCCAGGATAGGTCTAAGAACTTCACTGAGATTTTATTAATTCACAATTAATCAAAAGATAGGTATTCATGATTCAAGCTGGCTATGTAAATACTTCTTTTCAGTGTCTTATTTTTCCCATTATAACTTTTTCTGTTTTTAACAGTTCCAGAAACGCCTAAGCCTAGTTGTGGGGATCCAGCTGCAAGAAAAACGTTAGTCTCTTGGCCTGAGCCTG(T/C)ATCTAAACCTGA(G/T)(T/C)CTGCATCTAAACCCCATGGATATGTTTTATGCTATAAGAACAATTCAGGTAATGTAAAATTCCACTAGGGAAACAAAATCAAGAATTTTTAAATGTTGTAATTATTGTTTTAGCCAGGGAGCTACAAGGCTTTTATGAAGGTGATGAGAGATTCATGTTTGAGTTTGCTTAGATGAACACGCATCTCATGGAGAAGAATAAATATCTAGATCATTTATTAAAAATAGTGGACAGGACTAGAGTCCTTTAGAGCAGGTTACTCATCAGGACATTCCTTTATCTCTGGGCTAAGGTGGGAATGTACTTAGCTCTCATAGAEdit

Twelve nucleotide differences between the a and b alleles have been identified.  This assay is designed to detect a T to C point mutation (indicated in red), however, additional nucleotide differences in close proximity have been indicated in green.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
12964 Fluorophore AAC GTT AGT CTC TTG GCC TGA GC
12965 Fluorophore CTT CTC CAT GAG ATG CGT GTT C
12966 TCT CTT GGC CTG AGC

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.00
MgCl2 2.00
dNTPS-kapa 0.20
12964 0.50
12965 0.50
Glycerol 5.00
Kapa 2G HS taq polym 0.01
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 65.0 -- -0.5 C per cycle decrease
4 68.0 --
5 -- repeat steps 2-4 for 10 cycles
6 94.0 --
7 60.0 --
8 72.0 --
9 -- repeat steps 6-8 for 28 cycles
10 72.0 --
11 10.0 -- hold
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

Stock Number Strain Name
007594 B6.129P2-Ptprca Ightm1Mnz/J
006447 B6.129S6(CBA)-Cebpatm1Dgt/J
002014 B6.SJL-Ptprca Pepcb/BoyJ
018869 C.129S4(Cg)-Il13tm2.1Lky/J
4 strains use this protocol