Protocol 19118: Standard PCR Assay - Thpo<tm1.1(TPO)Flv>-Alternate 1
Version 4.0

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 = 273 bp
Heterozygote =273 bp and 460 bp
Wild type = 460 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
12143 CAG GAC TGA AAA GGG AAT CA Mutant A
12144 CGT TGG AAG GCC TTG AAT TT Mutant A
19391 ATG CCC TCT TCT TGA GCT TG Wild type A
19392 AGG CTT GGA GAA GGA GGA AG Wild type A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
12143 0.50 uM
12144 0.50 uM
19391 0.50 uM
19392 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
017712 C;129S4-Rag2tm1.1Flv Csf1tm1(CSF1)Flv Csf2/Il3tm1.1(CSF2,IL3)Flv Thpotm1.1(TPO)Flv Il2rgtm1.1Flv Tg(SIRPA)1Flv/J
017711 C;129S4-Rag2tm1.1Flv Csf1tm1(CSF1)Flv Csf2/Il3tm1.1(CSF2,IL3)Flv Thpotm1.1(TPO)Flv Il2rgtm1.1Flv/J
014594 C;129S4-Rag2tm1.1Flv Thpotm1.1(TPO)Flv Il2rgtm1.1Flv/J
3 strains use this protocol