Protocol 23543: Standard PCR Assay - Smn1tm6(SMN2)Mrph D Allele

FVB.Cg-Smn1^{tm6(SMN2)Mrph}/J

Stock No: 009381

Version 1.2

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 = 77
Heterozygote = 77 bp and 119 bp
Wild type = 119

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
oIMR9111		GTC CCT GGT CGA CAA GAA CAG		Wild type Forward	A
oIMR9112		ACG CTC TGC TGC TGA CTT AGG		Wild type Reverse	A
oIMR9289		GAT GGC TTT CTG ATG CTA CAA CAC		Mutant Forward	A
oIMR9290		CCA ATA TTC ACA GAG GCC AAC TG		Mutant Reverse	A

Reaction A

Component	Final Concentration
ddH2O
Kapa 2G HS buffer	1.30 X
MgCl2	2.60 mM
dNTP KAPA	0.26 mM
oIMR9111	0.50 uM
oIMR9112	0.50 uM
oIMR9289	0.50 uM
oIMR9290	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
009378	B6.129-Smn1^tm6(SMN2)Mrph/J
008704	B6;129-Smn1^tm6(SMN2)Mrph/J
009381	FVB.Cg-Smn1^tm6(SMN2)Mrph/J
3 strains use this protocol