Protocol 31801: Probe Assay - Ttll7<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

Mutant=  99 bp

Wild Type = 140 bp

>chr3:146896888+146897027 140bp TAAGCCCGTGTCATTTCCA CTTTGTCCACTAAGACAATCCATTC

Sequence

Sequence (deleted region in lower case)

TGTTGGGGAAGCCCTGAGGGTGAATGTACCTGTGTAGAGTTATATCAGCTGTCTCTGTGTTTTTACTGTACTAAAGTCACTCTCTAAACCctctgCAAGTGCTGTGTTTTGGGAAATTGCGCAGGACTATGACTAAGGCTTTTTGGTGCAACCTAATTCAGTTCCCAGCTTCTGTGGAGAGCAATTTGGTAAAAGTGCCGTCACTGGTAATGAGCTTATGCTATAAGGAGTGGggccagtctgcacctggaacactgtgggtatacgcatgtgcatttccattgtgaatgtctttaaggtgttctggctcaggtttgatgtaatcgtctcttttcagttcgtttagtaatagatgagatgggatttatgaaaactcctgatgaagatgagacaagcaatcttatctggtgtgatgctgctgttcagcaggagaagattacagacctgcagaactaccaggtggggactgacagtcagcatcggggagaaagtcacaccacttccaccacttccgtcatggttttatttagcttcttaatctgtccacatattagctttcagttctgagttccctgggccgcctgtctcgttgctcgctggctgtgctgactctttgcattaagcccgtgtcatttccaaactcaacctttagcttttttctttaaagatggatcaacagggaaaaattctaagttgatgctttgagttatgtacaccttaagatgagtgacagGGAATGGATTGTCTTAGTGGACAAAGACCTTATAGCTTCAGCTCCATTAGTCTGACTTTTACATCTATAGTGCAGTACAAATGTGTTTAGAAGAAATGGAAAGCAAATTGAAAAACCTACAAGGGATCCAAAGCAATCGATGTTTGCTACTTACCCTCCCTCCCTTCTTCCCCTTCCTCTCTCCTCCTCCCTCCCTCCCTCTCTCCTTCCCTCCTTTCCTCCCTCCCTTCCTCCCTCCTTCTCTCCTTCCTTCCCTCCCCTTTCCTTCCTCTCCCCTCCTTCCC

500 bp deletion beginning at Chromosome 3 positive strand position 146,896,502 bp and ending after 146,897,001 bp (GRCm38/mm10).  In addition there is a 5 bp deletion (CTCTG) 138 bp before the 500 bp deletion

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37693 GTT CCC AGC TTC TGT GGA GA Mutant Forward A
37694 CTT TGT CCA CTA AGA CAA TCC ATT C Common A
37695 TAA GCC CGT GTC ATT TCC A Wild type Forward A
37696 Fluorophore-1 AGT GCC GTC ACT GGT AAT GA Quencher-1 MUT Probe
37697 Fluorophore-2 TGG ATC AAC AGG GAA AAA TTC T Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37693 0.40 uM
37694 0.40 uM
37695 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.