Protocol 19539: Standard PCR Assay - Generic Rosa26Sor(GFP)
Version 2.0

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 = 384 bp
Heterozygote = 384 bp and 142 bp
Wild type = 142 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
21306 CTG GCT TCT GAG GAC CG Wild type Forward A
24500 CAG GAC AAC GCC CAC ACA Wild type Reverse A
24951 AGG GCG AGG AGC TGT TCA Mutant Forward A GFP F
24952 TGA AGT CGA TGC CCT TCA G Mutant Reverse A GFP R

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
21306 0.50 uM
24500 0.50 uM
24951 0.50 uM
24952 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
013205 129S-Gt(ROSA)26Sortm1(NOTCH3)Sat/Mmjax
013207 129S-Gt(ROSA)26Sortm2(NOTCH3*C455R)Sat/Mmjax
006148 B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J
005670 B6.Cg-Gt(ROSA)26Sortm1(rtTA,EGFP)Nagy/J
013206 B6;129S-Gt(ROSA)26Sortm1(NOTCH3*R1031C)Sat/Mmjax
018422 B6;FVB-Tg(Aicda-cre)1Rcas/J
031010 STOCK Gt(ROSA)26Sortm1(rtTA,EGFP)Nagy TgTn(pb-tetO-Pou5f1,-Klf4,-Myc,-Sox2,-mCherry)250Nagy Tg(Pou5f1-EGFP)1Nagy/J
005572 STOCK Gt(ROSA)26Sortm1(rtTA,EGFP)Nagy/J
8 strains use this protocol