Protocol 21168: Standard PCR Assay - Generic Rosa<#m1.1(CAG-CAS9)>
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 (28555, 28551) (Rsky) = 172 bp
Mutant (30936, 26179 and 24858) (Fezh, Dvs, & Mvw) = 290 bp
Wild type = 109 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
24493 AGC CTG CCC AGA AGA CTC C Wild type Reverse A
26932 CTC CGT CGT GGT CCT TAT AGT Mutant Reverse A
oIMR5984 GCT AAC CAT GTT CAT GCC TTC Mutant Forward A

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
24493 0.50 uM
26932 0.50 uM
oIMR5984 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
028555 B6(C)-Gt(ROSA)26Sorem1.1(CAG-cas9*,-EGFP)Rsky/J
026179 B6J.129(Cg)-Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP)Fezh/J
026430 B6N.129(Cg)-Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP)Fezh/J
028551 C57BL/6N-Gt(ROSA)26Sorem1(CAG-cas9*,-EGFP)Rsky/J
026558 FVB.129(B6)-Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP)Fezh/J
026557 NOD.129(Cg)-Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP)Fezh/J
030936 NOD/ShiLtJ-Gt(ROSA)26Sorem1.1(CAG-cas9*,-EGFP)Dvs/J
024858 STOCK Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP)Fezh/J
8 strains use this protocol