Protocol 23949: Standard PCR Assay - Smn1<tm5(Smn1/SMN2)Mrph> Alternate
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 = ~71 bp
Heterozygote = ~71 bp and 119 bp
Wild type = 119 bp
Separated by gel electrophoresis on a 3.0% agarose gel.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
11987 GGA TGC CAC CAT AGC ATT TTG Mutant Forward A
11988 CGG TCC TAA GGT AGC GAG TGA Mutant Reverse A
oIMR9111 GTC CCT GGT CGA CAA GAA CAG Wild type Forward A
oIMR9112 ACG CTC TGC TGC TGA CTT AGG Wild type Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
11987 0.50 uM
11988 0.50 uM
oIMR9111 0.50 uM
oIMR9112 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
033506 B6.Cg-Tia1tm1Andp Smn1tm5(Smn1/SMN2)Mrph/RnsMmjax
014561 FVB.Cg-Grm7Tg(SMN2)89Ahmb Smn1tm1Msd/Smn1tm5(Smn1/SMN2)Mrph/J
2 strains use this protocol