Protocol 22285: Standard PCR Assay - Gt(ROSA)26Sor<tm1Sor>
Version 1.3

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 = 300 bp
Heterozygote = 300 bp and 603 bp
Wild type = 603 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
oIMR8052 GCG AAG AGT TTG TCC TCA ACC Mutant A
oIMR8545 AAA GTC GCT CTG AGT TGT TAT Common A
oIMR8546 GGA GCG GGA GAA ATG GAT ATG Wild type Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR8052 0.50 uM
oIMR8545 0.50 uM
oIMR8546 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
014602 B6.Cg-Gt(ROSA)26Sortm1(rtTA*M2)Jae Col1a1tm1(tetO-mCherry)Eggn/J
023749 B6.Cg-Gt(ROSA)26Sortm1(rtTA*M2)Jae Tg(tetO-Pou5f1,-Sox2,-Klf4,-Myc)1Srn/J
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
007900 C57BL/6-Gt(ROSA)26Sortm1(HBEGF)Awai/J
12 strains use this protocol