Stock No: 014093
Protocol 14946: Pyrosequencing Assay - ** human CHRM3*Y149C
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 = C/C
Wild type has no PCR product for pyrosequencing.
 
GCCATTGACTa/gCGTAGCCAG
 
Sequencing primer anneals to the reverse strand.

Sequence

 

TCATCGGCAACATCCTGGTAATTGTGTCATTTAAGGTCAACAAGCAGCTGAAG
ACGGTCAACAACTACTTCCTCTTAAGCCTGGCCTGTGCCGATCTGATTATCGGG
GTCATTTCAATGAATCTGTTTACGACCTACATCATCATGAATCGATGGGCCTTAG
GGAACTTGGCCTGTGACCTCTGGCTTGCCATTGACT(a/g)CGTAGCCAGCAATGCC
TCTGTTATGAATCTTCTGGTCATCAGCTTTGACAGATACTTTTCCATCACGAGG
CCGCTCACGTACCGAGCCAAACGAACAACAAAGAGAGCCGGTGTGATGATCG
GTCTGGCTTGGGTCATCTCCTTTGTCCTTTGGGCTCCTGCCATCTTGTTCTGGC
AATACTTTGTTGGAAAGAGAACTGTGCCTCC

 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
13596 Fluorophore ATC ATG AAT CGA TGG GCC TTA Forward
13597 TCA AAG CTG ATG ACC AGA AGA TT Reverse
13598 GAG GCA TTG CTG GCT SEQ

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.00
MgCl2 2.00
dNTPS-kapa 0.20
13596 0.50
13597 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.