Genotyping Protocols Database
Stock No: 004861
Protocol 22421: Standard PCR Assay - Generic Pde6b
Version 1.3

Notes

This assay tests for the presence of the retinal degeneration Pde6brd1 mutation.

The WT-specific primer, oIMR2094, sits on a known SNP identified as rs47440869, located at the nucleotide indicated in red:  ACC TGC ATG TGA ACC CAG TAT TCT ATC.  Information can be found at http://www.informatics.jax.org/snp/rs47440869.

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 = 560 bp
Heterozygote = 240 bp and 560 bp
Wild type= 240 bp
Separated by gel electrophoresis on a 1.5% agarose gel.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR2093 AAG CTA GCT GCA GTA ACG CCA TTT Mutant A
oIMR2094 ACC TGC ATG TGA ACC CAG TAT TCT ATC Wild type A
oIMR2095 CTA CAG CCC CTC TCC AAG GTT TAT AG Common A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR2093 0.50 uM
oIMR2094 0.50 uM
oIMR2095 0.50 uM
Glycerol 6.50 %
Dye 1.00 X
Kapa 2G HS taq polym 0.03 U/ul
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 (Touchdown)
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.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.

Strains Using This Protocol

Stock Number Strain Name
001924 B6EiC3Sn a/A-Ts(1716)65Dn/J
004828 FVB.129P2-Pde6b+ Tyrc-ch/AntJ
004850 B6EiC3Sn-Rb(12.Ts171665Dn)2Cje/CjeDnJ
004861 B6EiC3Sn-Ts(16C-tel)1Cje/DnJ
4 strains use this protocol