Protocol 33142: Probe Assay - Gt(Rosa)26Sor(CAG) alternate 2

STOCK Gt(ROSA)26Sor^{tm1.2(CAG-EGFP)Fsh}/Mmjax

Stock No: 010812

Version 3.0

Notes

The size of the mutant band can vary between different Gt(Rosa)26sor(CAG) strains.

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

>chr6:113025964-113026059 96bp CTTCCCTCGTGATCTGCAAC CAGGACAACGCCCACACA

Mutant=  140 bp

Wild Type = 96 bp

Fam=Mut

Hex=Wt

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction	Note
17062		GTT ATG TAA CGC GGA ACT CCA		Mutant Reverse	A
24500		CAG GAC AAC GCC CAC ACA		Wild type Reverse	A
36158		CTT CCC TCG TGA TCT GCA AC		Common	A
39816	Fluorophore-1	ACG GGG TCA TTA GTT CAT AGC CCA	Quencher-1	MUT Probe
40263	Fluorophore-2	CCA GTC TTT CTA GAA GAT GGG CGG	Quencher-2	WT Probe

Reaction A

Component	Final Concentration
Kapa Probe Fast QPCR	1.00 X
ddH2O
17062	0.40 uM
24500	0.40 uM
36158	0.40 uM
Wt Probe	0.15 uM
Mutant Probe	0.15 uM
DNA

Cycling

Step	Temp °C	Time	Note
1	95.0	--
2	95.0	--
3	60.0	--
4		--	repeat steps 2-3 for 40 cycles
5	40.0	--	Forever

JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

Stock Number	Strain Name
039976	129(Cg)-Gt(ROSA)26Sor^tm5(CAG-Sun1/sfGFP)Nat/CdnyJ
006053	129-Gt(ROSA)26Sor^tm1(CAG-EGFP)Luo/J
006067	129-Gt(ROSA)26Sor^tm2(CAG-Dsred2/EGFP)Luo/J
006041	129-Gt(ROSA)26Sor^tm3(CAG-EGFP/Dsred2)Luo/J
007676	B6.129(Cg)-Gt(ROSA)26Sor^tm4(ACTB-tdTomato,-EGFP)Luo/J
006071	B6.129-Gt(ROSA)26Sor^tm1(CAG-EGFP)Luo/J
006080	B6.129-Gt(ROSA)26Sor^tm2(CAG-Dsred2/EGFP)Luo/J
006075	B6.129-Gt(ROSA)26Sor^tm3(CAG-EGFP/Dsred2)Luo/J
022367	B6.129S4-Gt(ROSA)26Sor^tm1(CAG-EGFP/Rpl10a,-birA)Wtp/J
017626	B6.Cg-Gt(ROSA)26Sor^tm1(CAG-GFP/Eif2c2)Zjh/J
040470	B6.Cg-Gt(ROSA)26Sor^tm1(CAG-Naaa)Dpio/J
026294	B6.Cg-Gt(ROSA)26Sor^tm2.2Ksvo/J
017455	B6;129-Gt(ROSA)26Sor^tm1(CAG-COP4*E123T*H134R,-tdTomato)Gfng/J
021429	B6;129-Gt(ROSA)26Sor^tm4(CAG-EGFP*)Nat/J
021039	B6;129-Gt(ROSA)26Sor^tm5(CAG-Sun1/sfGFP)Nat/J
026006	B6;129-Gt(ROSA)26Sor^tm7(CAG-tdTomato*)Nat/J
026005	B6;129-Gt(ROSA)26Sor^tm9(CAG-GFP*)Nat/J
010523	B6;129P2-Gt(ROSA)26Sor^tm1(CAG-ALPP)Fawa/J
024846	B6;129S-Gt(ROSA)26Sor^tm2.1Ksvo/J
023035	B6;129S6-Gt(ROSA)26Sor^tm1(CAG-tdTomato*,-EGFP*)Ees/J
036272	B6;CBA-Gt(ROSA)26Sor^em1(CAG-Ikzf2,-tdTomato)Zyliu/J
026556	B6N.129(B6J)-Gt(ROSA)26Sor^tm1(CAG-cas9*,-EGFP)Fezh/J
026430	B6N.129(Cg)-Gt(ROSA)26Sor^tm1.1(CAG-cas9*,-EGFP)Fezh/J
023537	B6N.129S6-Gt(ROSA)26Sor^tm1(CAG-tdTomato*,-EGFP*)Ees/J
031928	C57BL/6N-Gt(ROSA)26Sor^tm1(CAG-Il11)Cook/J
026862	D2.129(Cg)-Gt(ROSA)26Sor^tm4(ACTB-tdTomato,-EGFP)Luo/SjJ
026481	FVB.129(B6)-Gt(ROSA)26Sor^tm1(CAG-cas9*,-EGFP)Fezh/J
026558	FVB.129(B6)-Gt(ROSA)26Sor^tm1.1(CAG-cas9*,-EGFP)Fezh/J
026431	NOD.129(B6)-Gt(ROSA)26Sor^tm1(CAG-cas9*,-EGFP)Fezh/J
026557	NOD.129(Cg)-Gt(ROSA)26Sor^tm1.1(CAG-cas9*,-EGFP)Fezh/J
037456	NOD.129(Cg)-Gt(ROSA)26Sor^tm4(ACTB-tdTomato,-EGFP)Luo/YgchJ
039294	STOCK Gt(ROSA)26Sor^em4(CAG-Tbx2,-tdTomato)Zyliu/J
010675	STOCK Gt(ROSA)26Sor^tm1(CAG-EGFP)Fsh/Mmjax
010701	STOCK Gt(ROSA)26Sor^tm1.1(CAG-EGFP)Fsh/Mmjax
022386	STOCK Gt(ROSA)26Sor^tm1.1(CAG-EGFP/Rpl10a,-birA)Wtp/J
010812	STOCK Gt(ROSA)26Sor^tm1.2(CAG-EGFP)Fsh/Mmjax
018906	STOCK Gt(ROSA)26Sor^tm3(CAG-flpo/ERT2)Alj/J
007576	STOCK Gt(ROSA)26Sor^tm4(ACTB-tdTomato,-EGFP)Luo/J
024107	STOCK Gt(ROSA)26Sor^tm5(ACTB-tTA)Luo Igs7^tm93.1(tetO-GCaMP6f)Hze/HzeJ
024112	STOCK Gt(ROSA)26Sor^tm5(ACTB-tTA)Luo Igs7^tm94.1(tetO-GCaMP6s)Hze/J
014552	STOCK Nkx2-1^tm1.1(cre/ERT2)Zjh/J
41 strains use this protocol