Stock No: 004939
Protocol 13789: Pyrosequencing Assay - Lepr<db-5J>
Version 4.0

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

Mutant = T/T
Heterozygote = G/T
Wild type = G/G

Sequence

CCATTCCTCTGTTGAGGGGCATCTGGGTTCTTTCCAGCTCCTGGCTATTATAAATAAGGC
TGCTATGAACATAGTGGAGCATGTGTCCTTCTTACCGGTTGGGACATCTTCTGGATATAT
GCCCAGGAGAGGTATTGCTGGATCCTCCGGTAGTACTATGTCCAATTTTCTGAGGAACC
GCCAGACTGAGGAACCATAGATGCGAGCATCAGCAACAGAATACAAGAGACTGAAGA
CATTTCTCTCGAGAACATGTGTCTGACTCAGGCTTCTTATTGTTTTTCAGTTCCTATGAG
AG(g/t)GCCTGAATTTTGGAGAAAAATGGATGGGGACGTTACTAAAAAGGAGAGAAATG
TCACCTTGCTTTGGAAGGTATTTCTAATTTCTACCTTTTATTTTATTCTGAACTTATGTTTG
TCTCTACTTTGTTCATAGCCCTGTCATTACCTCAGAAATGACCCTCAGATCTTATGTTTTA
ATGCGTTTTGGGGATGTGGTTGTCAGAGCTGACTATCTGGAGTATCGACATTGTCAGGG
GCCTTTATTATTTTCTCGGTTCAATTATAATTGTGCCGTTGCAGTTTTGGCAAAGTCTTGA
ACTCTTTGGACATCACAATTTGTCATCTGTATCAGGGTTAAGATTACTAAGTGATAA

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
pIMR129 TTT TTC AGT TCC TAT GAG AG
poIMR0127 GTG TCT GAC TCA GGC TTC TTA TTG
poIMR0128 Fluorophore CTT TTT AGT AAC GTC CCC ATC CA

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.00
MgCl2 2.00
dNTPS-kapa 0.20
poIMR0127 0.50
poIMR0128 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

This is the only strain that uses this protocol.