Stock No: 014093
Protocol 15010: Pyrosequencing Assay - ** human CHRM3*A239G
Version 2.0

Notes

This assay will NOT distinguish hemizygous from homozygous trangenic animals.  This assay only confirms the point mutation.

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 = G/G
Wild type has no PCR product for pyrosequencing.
 
GCCATCGCTGc/gTTTTTATATGC
 
Sequencing primer anneal to the forward stand.

Sequence

 

CAGCTTTGACAGATACTTTTCCATCACGAGGCCGCTCACGTACCGAGCCAAACG
AACAACAAAGAGAGCCGGTGTGATGATCGGTCTGGCTTGGGTCATCTCCTTTGT
CCTTTGGGCTCCTGCCATCTTGTTCTGGCAATACTTTGTTGGAAAGAGAACTGT
GCCTCCGGGAGAGTGCTTCATTCAGTTCCTCAGTGAGCCCACCATTACTTTTGG
CACAGCCATCGCTG(c/g)TTTTTATATGCCTGTCACCATTATGACTATTTTATACTGGA
GGATCTATAAGGAAACTGAAAAGCGTACCAAAGAGCTTGCTGGCCTGCAAGCC
TCTGGGACAGAGGCAGAGACAGAAAACTTTGTCCACCCCACGGGCAGTTCTC
GAAGCTGCAGCAGTTACGAACTTCAACAG

 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
13599 CGG GAG AGT GCT TCA TTC AGT TC Forward
13600 Fluorophore CCG TGG GGT GGA CAA AGT TT Reverse
13601 TTG GCA CAG CCA TCG SEQ

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.00
MgCl2 2.00
dNTPS-kapa 0.20
13599 0.50
13600 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.