Stock No: 013122
Protocol 29734: Standard PCR Assay - Smc1a<tm1Mbk> STD PCR
Version 2.2

Notes

This assay does not work well without the use of a Hotstart Taq polymerase.

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= 480 bp
heterozygote = 480 bp & 380 bp
Wild type = 380 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
10927 GGT AGT GAG GGG TGG ACA GG A
10928 CTC TCG AGC ATA GAT ACT GG A
11185 GAG GGC CTC TCG AGC ATA GA A
11191 CGT GCC ATC TAG CAA AGG AG A
11192 GCC AGC TGA GAA GAT GCT G A
oIMR9041 CTT CGT ATA ATG TAT GCT ATA CG A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
10927 0.50 uM
10928 0.50 uM
11185 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

This is the only strain that uses this protocol.