Protocol 24971: Separated PCR Assay - Smn1<tm1Msd>

FVB.Cg-Grm7^{Tg(SMN2)89Ahmb} Smn1^tm1Msd Tg(SMN1*A2G)2023Ahmb/2J

Stock No: 007968

Version 7.2

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 = 500 bp
Heterozygote = 500 bp and 420 bp
Wild type = 420 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
oIMR3439		TTT TCT CCC TCT TCA GAG TGA T		Wild type Forward	B
oIMR3440		CTG TTT CAA GGG AGT TGT GGC		Wild type Reverse	B
oIMR7208		CTC CGG GAT ATT GGG ATT G		Mutant Forward	A
oIMR7210		GGT AAC GCC AGG GTT TTC C		Mutant Reverse	A

Reaction A

Component	Final Concentration
ddH2O
Kapa 2G HS buffer	1.30 X
MgCl2	2.60 mM
dNTPS-kapa	0.26 mM
oIMR7208	0.50 uM
oIMR7210	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
oIMR3439	0.50 uM
oIMR3440	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
007222	B6.Cg-Grm7^Tg(SMN2)89Ahmb Smn1^tm1Msd Tg(SMN1*A2G)2023Ahmb/J
006964	B6.Cg-Grm7^Tg(SMN2)89Ahmb Smn1^tm1Msd Tg(SMN2*delta7)4299Ahmb/J
006773	B6.Cg-Grm7^Tg(SMN2)89Ahmb Smn1^tm1Msd/J
008629	B6.Cg-Tg(SMN2)11Tro Smn1^tm1Msd/J
008631	B6.Cg-Tg(SMN2)11Tro Tg(SMN2)46Tro Smn1^tm1Msd/J
008630	B6.Cg-Tg(SMN2)46Tro Smn1^tm1Msd/J
008209	FVB.Cg-Grm7^Tg(SMN2)89Ahmb Smn1^tm1Msd Tg(ACTA1-SMN)69Ahmb/J
007968	FVB.Cg-Grm7^Tg(SMN2)89Ahmb Smn1^tm1Msd Tg(SMN1*A2G)2023Ahmb/2J
005026	FVB.Cg-Grm7^Tg(SMN2)89Ahmb Smn1^tm1Msd Tg(SMN1*A2G)2023Ahmb/J
007952	FVB.Cg-Grm7^Tg(SMN2)89Ahmb Smn1^tm1Msd Tg(SMN2*delta7)4299Ahmb/2J
005025	FVB.Cg-Grm7^Tg(SMN2)89Ahmb Smn1^tm1Msd Tg(SMN2*delta7)4299Ahmb/J
007949	FVB.Cg-Grm7^Tg(SMN2)89Ahmb Smn1^tm1Msd/2J
005024	FVB.Cg-Grm7^Tg(SMN2)89Ahmb Smn1^tm1Msd/J
008206	FVB.Cg-Smn1^tm1Msd Tg(SMN2)566Ahmb/J
008203	STOCK Grm7^Tg(SMN2)89Ahmb Smn1^tm1Msd Tg(ACTA1-SMN)63Ahmb/J
006553	STOCK Grm7^Tg(SMN2)89Ahmb Smn1^tm1Msd Tg(H2-K1-tsA58)6Kio Tg(SMN2*delta7)4299Ahmb/J
006570	STOCK Grm7^Tg(SMN2)89Ahmb Smn1^tm1Msd Tg(Hlxb9-GFP)1Tmj/J
008212	STOCK Grm7^Tg(SMN2)89Ahmb Smn1^tm1Msd Tg(Prnp-SMN)92Ahmb/J
017596	STOCK Gt(ROSA)26Sor^tm1.1(rtTA,EGFP)Nagy Grm7^Tg(SMN2)89Ahmb Smn1^tm1Msd Tg(SMN2*delta7)4299Ahmb Tg(tetO-SMN2,-luc)#aAhmb/J
017597	STOCK Gt(ROSA)26Sor^tm1.1(rtTA,EGFP)Nagy Grm7^Tg(SMN2)89Ahmb Smn1^tm1Msd Tg(SMN2*delta7)4299Ahmb Tg(tetO-SMN2,-luc)#bAhmb/J
007022	STOCK Mnx1^tm4(cre)Tmj Grm7^Tg(SMN2)89Ahmb Smn1^tm1Msd Tg(SMN2*delta7)4299Ahmb/J
21 strains use this protocol