Protocol 36102: Probe Assay - Tmem147<em1(IMPC)J>
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.

 

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

>chr7:30727524-30727667 144bp AATCCTCGGACTTAGGTCTCAC TCTAGTGTGTTCATTCCTTAGCTTG

Mut= 158 bp

Wt= 144 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GAGACAACGCTTAGGGACTATGCCCGAATACCTTAGAAGGGATGCAAGGCCAGAAAGGCCCTGATGCAACTTTTTCTTACAACTGTTGTAAAATGAGAAAACCTCTTAGTGCGATATTTCACCCACAGGGTGTCGCAGGGTGCTGCCTAGGCATTTTTAGACATCAGATTCCCCcgtgaacttctagacttttagaaaccaggccacttgccctctatctcccaagatcacgtgatcttgccacttacacctagaatgacccctagaacccagcgtcatatcctctacttgcacacttactttccacagtatccacatagttttttgtttgtttgtttcggtttttaatctccctcaccttccagtgacctttctactttctgatctcagatgttgttcttggccactttcttccccacctgggaaggtggcatctatgacttcattggggtaagttgaacaagggaagggcaaggaagagtgcaggagtcggttcacctggtccccgtgtttactcgagcctctacctgacccacaggaattcatgaaggccagtgtggatgtggcagacctgataggcctaaaccttgtcatgtctcggaatgcaggcaagggggagtacaagattatggttgctgccctgggctgggccactgctgaactcattatgtcccggtgtgtggagcaaactggagcccagacttccgggaatggacagctgggtcccacaggggtgctgaggggaggtgatcagaggctgggtgctgggggtgggggagtctgggtactagagactgcctccttttgccttcctcagatgcatccccctctgggtgggagcccgaggcattgaatttgactggaaatatatccagatgagcattgactccaacatcagtctggtatgcagtctagccctagcctacctttatgttgttagccatacttctcccaaaaacttggctccaacttcccagtcccttcctgcctccctgcaggtccactacatcgttgcatcagctcaggtctggatgataacacgctatgacctgtaccacaccttccggcctgctgttctcctcctgatgttccttagtgtctacaaggcctttgtcatggagtgagctgggtggggtatgaggctgggttcaaatgggggtgtgttttctctctccttttcctcattgtgatgtttccccacaggaccttcgtccacctcttctccttgggtagttggacagcactgctcgcccgggcagtagtaacaggactgctggccctcagcaccttggccctgtatgttgctgttgtcaatgtacactcctagcttgacatctcaggtgttggcgaaccttttcatttcctctctccagatttcagtgaagtaaacagtgtttggaaaggtttttgttatgtccatctctctctctcagaaaccgaatcctcggacttaggtctcacagagtctgtctggcttcagaaccagataaataagctggaatgttggccagtcttcctggtttccccatgagaacctgggggcTTGGTAATCATTGAACAAGCTAAGGAATGAACACACTAGAGTTCAGGAGGGGGCTTTGGGGTAGAGGGGCATTCTTCCTCAACTTACAGCTTGGTTTGAGCCCTCTTCCATGGTGAGTCTTCCCAAAACTGGTTACCTCCAACTTTGCAACCCCCCATTTCTCCACTCTAGTCCATCCACCATGTAACAGCTAGGG

This  mutation is a 1369 bp deletion beginning at Chromosome 7 position 30,727,564 bp and ending after 30,728,932 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
47837 AAT CCT CGG ACT TAG GTC TCA C Wild type Forward A
47838 TCT AGT GTG TTC ATT CCT TAG CTT G Common A
47839 GGC CCT GAT GCA ACT TTT TC Mutant Forward A
47840 Fluorophore-1 AGC TGG AAT GTT GGC CAG T Quencher-1 WT Probe
47841 Fluorophore-2 CCC ACA GGG TGT CGC A Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
47837 0.40 uM
47838 0.40 uM
47839 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.