Stock No: 002217
Protocol 23572: Standard PCR Assay - Selp<tm1Hyn>
Version 2.2

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

Mutant = 479 bp
Heterozygote = 322 bp and 479 bp
Wild type = 322 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
oIMR0295 TTG TAA ATC AGA AGG AAG TGG Wild type Reverse A
oIMR0296 AGA GTT ACT CTT GAT GTA GAT CTC C Common A, B
oIMR2060 CAC GAG ACT AGT GAG ACG TG Mutant Reverse B

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTPS-kapa 0.26 mM
oIMR0295 0.50 uM
oIMR0296 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.

Reaction B

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTPS-kapa 0.26 mM
oIMR0296 0.50 uM
oIMR2060 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
008437 B6.129S2-Seletm1Hyn Selptm1Hyn/J
008441 B6.129S2-Selltm3Hyn Selptm1Hyn/J
008432 B6.129S2-Selptm1Hyn/J
003807 B6;129S-Seletm1Hyn Selltm1Hyn Selptm1Hyn/J
003805 B6;129S-Selltm3Hyn Selptm1Hyn/J
002916 B6;129S2-Seletm1Hyn Selptm1Hyn/J
002217 B6;129S2-Selptm1Hyn/J
008438 C.129S2(B6)-Seletm1Hyn Selptm1Hyn/J
008442 C.129S2(B6)-Selltm3Hyn Selptm1Hyn/J
008433 C.129S2(B6)-Selptm1Hyn/J
10 strains use this protocol