Protocol 13514: Pyrosequencing Assay - Plp1<jp>
Version 2.0

Notes

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

Sequence

GGCTGTTCTCACTTTTTCTCCGATTGACTGATATTAACAACGCAAAGCAGCACAT
TTCAATAATATAAAACTTCACAATTAGTCAATAGCACCTTTGTTCTATGTAACTTTTT
AGAAAAAAAGATGATGCATACTGTCAATATCAGGTTTACAGCCAGGATAGAGAAG
GTAGAAAGCTATGTAGAACGTCTCCGCATGGCCTCTAGCCTTATGAAGTTTACTC
TGGCTGCTTTTATGTATCTTa/gGGTGTTCTCCCATGGAATGCTTTCCCTGGCAAGG
TTTGTGGCTCCAACCTTCTGTCCATCTGCAAAACAGCTGAGGTAAGTGAATGAG
AAGAGTGCTTTTTAAAAAATAGATTGGCTAGACATGGAGGAATTTCTACCTATAAT
AGAAGTTTCTCCTTATGAACTTAAGTTTTAAGGATTATATTATTTCCTTATTGGACT
TTGTATTAGCAGATTTCCCTTCTACAAAATAATGGTGAAATGTACTTGAATCAATGA
GCATACTTATTAAAAGTCAAAGTACTTATTAACCCCAGAGCTGCACTAGAAGTCTT
CCTTTCCCAAACTCCTCTCATTAAAAGAAACATGTGTAGCTTTAAGTAAAGATAAT
ACTTAGCAGTAAAATTTCCCAAATGAAAATTTTCATTT

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
9064 Fluorophore TAG AAC GTC TCC GCA TGG C
9065 ACA GAA GGT TGG AGC CAC AAA
9066 ATT CCA TGG GAG AAC AC

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.00
MgCl2 2.00
dNTPS-kapa 0.20
9064 0.50
9065 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.