Stock No: 013100
Protocol 14129: Pyrosequencing Assay - Prdm16<csp1>
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

REVERSE SEQ
WT=G
MUT=T

Sequence

CTATAGTGATTCCCATCCCCTGTACA(c/a)AGCTTGGAGCAACACATGATCGTCCAC

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
11112 Fluorophore AGG TAG AGT TTG CAC GCA TTC C
11113 CGT GTG GAC GAT CAT GTG TTG
11114 CAT GTG TTG CTC CAA G

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.00
MgCl2 2.00
dNTPS-kapa 0.20
11112 0.50
11113 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.