Protocol 36758: Probe Assay - Elp6<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

>chr9:110312428+110312539 112bp AGAGAGAGGGTGAGGAGGTG CAAATCCTCTTCCTACAACCAGA

Mutant=  93 bp

Wild Type = 112 bp

Sequence

Wt Sequence (deletions in lower case):

GGTTGCGTTTCCAGTATCATAGCCAGGGCTACATAGAGAAATCCCATCTCAAAAAACCAAAATAACAAAACAAAGCAAAACAAAAAATAGAGTTCAAAGTTGTCCTGGGTTATACAGGGAGTTGAAGACCAGCATGTGCTACCCGAGACACTGTGTCATAGAGAGAGGGTGAGGAGGTGCGCCAGAGTGCAAGGCctcggctaggccttcctcatagctcctgtctccagaggattgaaatagttatgtctggttgtaggaagaggatttgtttaaaaagtcatttattttgttttttagctaattgtaaagtctgctttgtggcacttgtccagtccttcagccattacaatattgtgggacagaaactggtaagtgtttcagccctctgtggtgacatcccctcaaaggagcatgtgtcacttgtggctaggactgggactttcttcttcttcttcttcttctccttctccttcttccttcttccttcttccttcttcttccttcttcttccttcttcttccttcttcttccttcttcttccttcttcttccttcttcttccttcttcttccttcttcttccttcttcttcttcttcttcttcttcttcttcttcttcttcttcttcttcttcttcttcttccttcttccttcttccttcttccttcttccttcttccttcttcttctttttttatttattattttttttattttttgagacagggtttctctgtgcagtcctgtcctggaactcactctgtagacccaagtaaacaaacaaacaaataaaacaaccaaacacaagctgggggtatgacttagtggtaggactgggtttaatccccagttctagaaaacaaaaggggaagaggacattttggcttggtatgatggttccaaggcctcaaactcagaaattcaccggcctctgcctcccaagtgctgggattacaggcatgtgccaccactgcctggctgaagctgggcttttctgcttaggttgtggagaacctaacaggaaggactgaggatattttgggactctgaaccccttatccaaaggatgggcgtgttttattgaaagcatatagaaccagacctagtatatgtagttgtcagtgactcaaacccttctcctgattgcggccagttaagattttcttgcctagctgagtatgttttcacatgactttaatcccagcactaggaggtagaggcaggacacaagatcggggctgggactgcagaggtggctccgtggttagcactgctgcagaggacctgagttaggtgcccagcacccccgtcaggcagcttacagcctcctgaggcagctgcacttgcatgcagagacatttacacataattaaaaagtaaacataacctcagcattcaggaagcagagacaggccggcctggtcacagatctagttccgggacagctagggatacacagacactctgtcttggggggaaaaaaaaggtcacagtcatgttctactgcattttgggtcaaagcttgcttgggaggatacccaagtccttgttgcaaatatcagatagcagagtggaaatagacaggaacacttaagcggggtttggagtatgtcttgtaccctcttattagctagtatgtagtttctctgaaaggctctccattccccagagggcacagcaattcactctgctctttgtcttgtctagggtgtcagcttgacagcggctcgggacagagggcagcttgtgttcctggagggtctcaagtcctctgtggaggtcctcttccactctcaggacgagcctcaccccctgcagtttctcaggtcagaaccatagtgctggggtggggcaggcaagggctgctgtgttgtgtgggaaggcagaagtgacttcagggatcagactctttccccgtgttcctgtgtgtcagatagcccctggctgccatcatgaagctgacggtgggtggtggtccccagagctgtgggaagcgtccgtcatttgcTGGGAGGATGGCGCGGTGATGTTACCCTGCTGTTTCGCTGATGGGGGCTTAGACCTCTCATGCACCTACTTTTGCTCTTTCTTGCCTTCATGTAGAAACTTCATCTTATGGGCCAGTGACCTTATTGGGACATAGCCATATTTGTTTGTCATTGATTGTCCCTGGAATATTGGGGGGGAAAATG

 

A 1813 bp deletion beginning at Chromosome 9 position 110,312,464 bp and ending after 110,314,276 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49602 AGA GAG AGG GTG AGG AGG TG Common A
49603 CAA ATC CTC TTC CTA CAA CCA GA Wild type Reverse A
49604 GAG GTC TAA GCC CCC ATC AG Mutant Reverse A
49605 Fluorophore-1 CGG CTA GGC CTT CCT CAT AG Quencher-1 WT Probe
49606 Fluorophore-2 TGC AAG GCT GGG AGG AT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49602 0.40 uM
49603 0.40 uM
49604 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.