Protocol 32240: Standard PCR Assay - Gt(ROSA)26Sor<tm1(rtTA*M2)Jae>

STOCK Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} Col1a1^{tm3(tetO-Pou5f1,-Sox2,-Klf4,-Myc)Jae}/J

Stock No: 011004

Version 1.0

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 241 bp
Wild type = 241 bp

>chr6:113025964-113026204 241bp AAGGGAGCTGCAGTGGAGTA CAGGACAACGCCCACACA

 

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction	Note
24500		CAG GAC AAC GCC CAC ACA		Wild type Reverse	A
31499		CTT GTT CTT CAC GTG CCA GT		Mutant Reverse	A
oIMR9020		AAG GGA GCT GCA GTG GAG TA		Common	A

Reaction A

Component	Final Concentration
ddH2O
Kapa 2G HS buffer	1.30 X
MgCl2	2.60 mM
dNTP KAPA	0.26 mM
24500	0.50 uM
31499	0.50 uM
oIMR9020	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
036019	B6.Cg-Gt(ROSA)26Sor^tm1(rtTA*M2)Jae Col1a1^tm1(tetO-EGFP,-MIR26A2)Jtm/J
037530	B6.Cg-Gt(ROSA)26Sor^tm1(rtTA*M2)Jae Col1a1^tm1(tetO-Plk4,-EYFP)Ahol/J
036020	B6.Cg-Gt(ROSA)26Sor^tm1(rtTA*M2)Jae Col1a1^tm1.1(tetO-EGFP,-MIR26A2)Jtm/J
034364	B6;129-Gt(ROSA)26Sor^tm1(rtTA*M2)Jae Col1a1^tm3(tetO-H3f3a)Hoch/J
034365	B6;129-Gt(ROSA)26Sor^tm1(rtTA*M2)Jae Col1a1^tm4(tetO-H3f3a*K36M)Hoch/J
034366	B6;129-Gt(ROSA)26Sor^tm1(rtTA*M2)Jae Col1a1^tm5(tetO-H3f3a*K9M)Hoch/J
035243	B6;129-Gt(ROSA)26Sor^tm1(rtTA*M2)Jae Hprt1^tm2(tetO-mediumFT*)Sguo/Mmjax
036998	B6;129S4-Gt(ROSA)26Sor^tm1(rtTA*M2)Jae Col1a1^tm1(tetO-cas9*)Jnxu/J
034380	B6;129S4-Gt(ROSA)26Sor^tm1(rtTA*M2)Jae Col1a1^tm1(tetO-GFP/RNAi:rluc)Begg/J
034150	B6;129S4-Gt(ROSA)26Sor^tm1(rtTA*M2)Jae Col1a1^tm2(tetO-GFP/RNAi:Rpl5)Begg/J
038749	B6;129S4-Gt(ROSA)26Sor^tm1(rtTA*M2)Jae Col1a1^tm5(tetO-Cas9/Dntt*)Fcam/J
034363	B6;129S4-Gt(ROSA)26Sor^tm1(rtTA*M2)Jae Col1a1^tm6(tetO-Nanog)Hoch/J
034917	B6;129S4-Gt(ROSA)26Sor^tm1(rtTA*M2)Jae Col1a1^tm7(tetO-Pou5f1,-Klf4,-Sox2,-mCherry)Hoch/J
035244	STOCK Gt(ROSA)26Sor^tm1(rtTA*M2)Jae Hprt1^tm3(tetO-slowFT*)Sguo/Mmjax
011004	STOCK Gt(ROSA)26Sor^tm1(rtTA*M2)Jae Col1a1^tm3(tetO-Pou5f1,-Sox2,-Klf4,-Myc)Jae/J
011011	STOCK Gt(ROSA)26Sor^tm1(rtTA*M2)Jae Col1a1^tm4(tetO-Pou5f1,-Sox2,-Klf4,-Myc)Jae/J
16 strains use this protocol