Protocol 37651: Probe Assay - Trim35<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

>chr14:66303913+66304015 103bp GCATTGAGTTGCTTTGCTGA GTCGTTTGCACCTACACCAG

Mut = 118 bp

Wt = 103 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

GGGTGGGTAGGGGGCAGTATTTAGCATTATAATACATAGCAAAAGACCAAACCTCAACATGGCTGGGCCAGGTAGGAAGCCTGGGAGGACCGAGAGGGGAGGGAGGATGAGTCCCTGGCATTGGCCCACCCATCTCTGTCTTTGGGGCTCTTCATGTCACACCTAGAGCATTGAGTTGCTTTGCTGAAAGTGGTCCCCAGctggggcgtcccaaagccattacatctgaccttgcttgcttctccctcctctggtgtaggtgcaaacgacttggctggaaggccgcatccgggatgagtttgacaagctccgtgatttcctgagggtggaggaacaggccaccttggatgccatgaaggaagagagcagaaagaagcacctgcaggctgaggagaagatgaagcagctagcagaacagaccgaggcgctggctcgggagattgagcgtctgcagatggagatgaaggaagatgacatgaccttcctcatggtaagaggctcatcgtgtgaactgagggtggggtcagggtcaggggccagcagctcagaccacagaccatgttcaacagtctttctgtgatggtggagggccttattggcatgccttgtggcagagcaaggcttggcggttgagaataccctcttaccttcacctccgccccttactgttgctgctttcttattctttgattggttatctctctatctctaatgaaatagtcaaattgtgtaataatttaagctttaaaatttttattacatttttatttatttactgtagatgagtgtcttagggttcatttactatgaagagacaccattaccacagcaactcttataaaggaaaatgcctaactggggctggcgtacagttcagaggtttaatccattgtcattatggtgggaagcatggcagcacacaagcagacatggtgctagagagggagttgagagttctacatctggatcagtaggcagcaggaagagagagtgagccactgggtctggcttgagcttcatccccctgtgacacacttcctccgagaaggccacacctcctatactgcccctctctatggacctatgggggccatgtcaaactgtctctatgggtacacattgcagagtgcctgtatggaagtcagtcggtcttcttttaccacgtgggtcccacagaccaaactgaagttgctaagcttgacagcaagcaccttaacctgctgagccatttagctggctccgttgttgttgttgtttgttttgtttatttgttgctttgttgtttgctttctcctctgtctttcttgcttttctccctccctccctccctccctccctcactgtgcagtgctggatctgtagcttaggctttgtgaatgacaagtactctcactgaggtatcttttatcttttccttagttttggggatgggtgtgagggtacaggattgcactctgccttctttctctaacgctttcaggctgaggagctggctcagcctgtgaagtaggaggagccagtctgatccctagatgccacagaaaataagctgagcatggtggctcactctgtttgtttttaagctgaaattattatacaatttgactattgcattcaggagttgagaacaaccaactaaccagccaacaaaaaaagccactaagtggcctgccagcctagcctgcttggtgcgatccagggcagtgaaacacactgtactaaagaacaaggcaggaataagctgtggtttacctctggccccatgagacgtatgtattaatgctgcgcgcgcgcgcacacacacacacacacacatgagtgcacaggaaagaaattctgttcttcctttaaaaacatgattcattgtctttcttccctttactgattttgtccttccttgcagagcagtttcccagacttgacagctagtttatcagtcagccccctttgatggcgagtgttcccctcgtgcatggcgtttttacctgtcagttggccttccgttctcacttgagttacttctgcttttgctgtttatgggcagtaagactctaagcgtgtgtgtactggatttgtgtggacatgaacttttcgatgtgtacccaagggatttctggattacttgataattctgctttaacccattaagggctgctaggcagttttaccaaagtagctacactattttgtcttcccaccagcagagtctgggtgtcaagttcctgtacctcttggccacttcttttctccatagctttccttgtgagtcacttgggctctcattgtgcttccagtgtgcactctctggatagctgatgacactgggcatctgtcttttcagtggctttgtggacattactagttcttgcttaaatgtctgtttaatttgtttatacagttttttccttcctttttttttttgttttggttttttttgtttgtttgtttgggttccccccacccccaagatggggtatctctgtgtaggtagccctggctgtcctggaatttactctgtaccaggctggcctcaaactcagaacacctgcctctgcctcccgagtgcaggaactagagatgtgtgctaacgtgcccagctatccagttttcagtagggttgtctgttcattgtttagctgtgagagttcgttccttttctgctccagtatgactcttacctcttccgtcttagatttgcatttaaatcttacatgtattttaagggtgttttgttttgtttttgtttttgttttgaatctgacacaaggcagaggctttccccattttggttgtattgacatgttctctgagaaggcagttctattaacttaccttggtcctgtagaaactccagcgttatctagactgtcacttctgtcctcattactcctgatatctctaccacagcagcttgtgtaacaagtgtgaagtttgggagtatgagtcctccaacttggtggttttcaaggttgcttttattcagcatttcctgaaactccatatgaactttaggatcagttttaagttttgccaaagaaggcccaaggccttgtgtataccgagcaaggtctccctcccatggctgcatcctcaccatgggactttcaatgggaggtgagggggggacactgaatctgagatgagatccccatggggtatgtgacagctggatgatgtcagttcttctcatccatgagtgtgggacatctggcaattcttggtcagaaattacctgcagtttcttagtctttgtctctgctttttttttttttctttccagaaacacaagagccgaaaacgccggtaagttggcaaacccaatggggatgggaaagggtaaatagatagaaagccaggccttcactgtccagtggtggaagggctaggcaggtagtgtgtccaactgggtggaatccagagggagctgtagaggccagccaaggggagcgctttaaagacatcccagttctggttggcatggagacagggagttgtttcagaacatagagggtcgatactctccacagGGATTGACAGCACCAGGCACATGACTCATGTTTCTCAGTCGTCAACAGGCTTGGTTTTTGGCTGGCTTCCTACCTCTGGGAAAGAGCGTTGGCATGCTTTGCTGTGGGTGGATGCTTGCTGGGCTCTCCAAGATGGGTA

This mutation is a 3327 bp deletion beginning at Chromosome 14 position 66,303,946 bp and ending after 66,307,272 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
51870 GCA TTG AGT TGC TTT GCT GA Common A
51871 GTC GTT TGC ACC TAC ACC AG Wild type Reverse A
51872 TCT TTC CCA GAG GTA GGA AGC Mutant Reverse A
51873 Fluorophore-1 CGT CCC AAA GCC ATT ACA TCT Quencher-1 WT Probe
51876 Fluorophore-2 CAG GGA TTG ACA GCA CCA GG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
51870 0.40 uM
51871 0.40 uM
51872 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.