Protocol 21931: End Point Analysis Assay - Col1a2<tm1.1Mcbr>-EP
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 =T/T
Heterozygote = G/T
Wild type = G/G

Sequence

attagtctcttccaaggtaactgcaaagcgtgaaaacaaccctctttctaccgctcttctctc
ttcctcccctcatctatgtgaggcccattaagcgactcaccactgctctttggtaccattaca
gGGTGAAGCTGGTGCTGCC(g/t)GTCCTTCTGGCCCAGCTGGTCCTCGGGGTAGCCCTgtaagt
agaaaccacagctttttggatattcatagttcatggtttacagacatctgagaggctgtgag
tgtgatgtctttatctcccacc

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
21325 TCT ACC GCT CTT CTC TCT TCC Forward A
21326 GTG GTT TCT ACT TAC AGG GCT AC Reverse A
21327 Fluorophore-1 GTG CTG CCG GTC CTT CT Quencher-1 WT Probe
21328 Fluorophore-2 GGT GCT GCC TGT CCT TCT G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
21325 0.40 uM
21326 0.40 uM
Wt Probe 0.15 uM
Mutant Probe 0.15 uM
DNA

Cycling

Step Temp °C Time Note
1 95.0 --
2 95.0 --
3 60.0 --
4 -- repeat steps 2-3 for 40 cycles
5 40.0 -- Forever
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

This is the only strain that uses this protocol.