Stock No: 009361
Protocol 3624: Pyrosequencing Assay - Btk<xid>
Version 1.0

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
Heterozygote T/C
Wild type = C

Sequence

The C to T transition point mutation at position 219 is predicted to change amino acid 28 from an arginine to a cysteine in the encoded protein. (J:13209)


GCTGCAGTGATACTGGAGAGCATCTTTCTGAAGCGCTCCCAGCAGAAAAAGAAAA
CATCACCTTTAAACTTCAAGAAG(c/t)GCCTGTTTCTCTTGACTGTACACAAACTTTCA
TACTATGAATATGACTTTGAACGTGGGGTAAGTTTCCCAAATGAG

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
pIMR326 ATC TTT CTG AAG CGC TCC CA
pIMR327 Fluorophore GGG AAA CTT ACC CCA CGT TCA A
pIMR328 TCA CCT TTA AAC TTC AAG A

Reaction A

Component Final Concentration
ddH2O
KAPA LR HotStart Buf Buffer does not contain MgCl2. 1.00
MgCl2 2.00
dNTP 0.13
KAPA LR HotStart Taq Initial Denature 3 Min This is a two enzyme system. One with proofreeading the other without. HotStart is provided by antibody. 0.02
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 60.0 --
4 65.0 --
5 -- Go to step 2-4, 50 times
6 65.0 --
7 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
009361 B6.CBA-Btkxid/AllmJ
001011 CBA/CaHN-Btkxid/J
2 strains use this protocol