Protocol 32240: Standard PCR Assay - Gt(ROSA)26Sor<tm1(rtTA*M2)Jae>
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
034364 B6;129-Gt(ROSA)26Sortm1(rtTA*M2)Jae Col1a1tm3(tetO-H3f3a)Hoch/J
034365 B6;129-Gt(ROSA)26Sortm1(rtTA*M2)Jae Col1a1tm4(tetO-H3f3a*K36M)Hoch/J
034366 B6;129-Gt(ROSA)26Sortm1(rtTA*M2)Jae Col1a1tm5(tetO-H3f3a*K9M)Hoch/J
034380 B6;129S4-Gt(ROSA)26Sortm1(rtTA*M2)Jae Col1a1tm1(tetO-GFP/RNAi:rluc)Beggs/J
034150 B6;129S4-Gt(ROSA)26Sortm1(rtTA*M2)Jae Col1a1tm2(tetO-GFP/RNAi:Rpl5)Beggs/J
034363 B6;129S4-Gt(ROSA)26Sortm1(rtTA*M2)Jae Col1a1tm6(tetO-Nanog)Hoch/J
034917 B6;129S4-Gt(ROSA)26Sortm1(rtTA*M2)Jae Col1a1tm7(tetO-Pou5f1,-Klf4,-Sox2,-mCherry)Hoch/J
035243 B6J.Cg-Gt(ROSA)26Sortm1(rtTA*M2)Jae Hprttm2(tetO-mediumFT*)Sguo/Mmjax
035244 B6J.Cg-Gt(ROSA)26Sortm1(rtTA*M2)Jae Hprttm3(tetO-slowFT*)Sguo/Mmjax
011004 STOCK Gt(ROSA)26Sortm1(rtTA*M2)Jae Col1a1tm3(tetO-Pou5f1,-Sox2,-Klf4,-Myc)Jae/J
011011 STOCK Gt(ROSA)26Sortm1(rtTA*M2)Jae Col1a1tm4(tetO-Pou5f1,-Sox2,-Klf4,-Myc)Jae/J
11 strains use this protocol