Stock No: 013158
Protocol 24119: Standard PCR Assay - Utrn<tm1Ked>alternate1
Version 3.2

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 = ~310 bp
Heterozygote = 349 bp and ~310 bp
Wild type = 349 bp

Two protocols are provided because this one requires a long run on 3% gel.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
10791 CGC TTC CTC GTG CTT TAC GGT AT Mutant Forward A Neo F
12402 AAG ATT TGC AGA CCG GAA GA Common A
14702 TGT CAT TCT CTG AGG CCT TTC Wild type Forward A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
10791 0.50 uM
12402 0.50 uM
14702 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
018802 B10ScSn.129(Cg)-Utrntm1Ked/J
019014 B10ScSn.Cg-Utrntm1Ked Dmdmdx/J
016904 D2.129(B6)-Utrntm1Ked/J
022506 D2.Cg-Utrntm1Ked Dmdmdx/J
014563 STOCK Utrntm1Ked Dmdmdx/J
013158 STOCK Utrntm1Ked/J
6 strains use this protocol