Stock No: 028549
Protocol 19883: Standard PCR Assay - Cbfb<tm1.1Ddg> Flag-Alternate 2
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 = ~110 bp
Heterozygote = ~110 bp and 185 bp
Wild type = 185 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
10223 GAC TAC AAA GAC GAT GAC GAC AAG Mutant Forward A
28702 GGT CAA GGC GGT CAG CAC Wild type Forward A
29424 CCT CCC CAA CCG CCT CAC Common A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
10223 0.50 uM
28702 0.50 uM
29424 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
028549 B6.129S6(SJL)-Cbfbtm1.1Ddg/J
028550 STOCK Cbfbtm2.1Ddg/J
2 strains use this protocol