Protocol 31913: Probe Assay - Mettl6<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

>chr14:31482590-31482689 100bp TAACACACAAGTCCCAAGTCC TCTACTCTGCGGGCCTCACT

Mut= 124 bp

Wt= 100 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence: (deleted region in lower case)

 

AACTGTTTTATGCTCCATCATATCTAAAAAAAGTGACTTCTAAGGCATTTTATTTACTTATTGGTTTTTTGAGACAGGATCCTTTTGCTAGGTAAACCAGGTAGGCTAGGTACTGTGTAGGTCCGGCTAACCTCAAACTCAAAGTGATATTCCTGCCTCCCTCTTGAACTCTGTGATTACGGTATGTACTCCACACCTGACATGGTTTTTTAAAGAGCAACCATTCATAGTAAGGGTATTTTGTATGCCTTCTTTCACCACTCCCACTggtctgtgttttaattcctaatgtcatcagattacattttagttgaccgggtttatacagcctgctttattacttttgtaggtattaaaaccaggcagaagtgtcttattccgtgactacgggctgaatgatcacgccatgcttagatttaaagctggaagcaaacttggagaaaatttttatgtcaggcaagatggaaccagatcgtatttttttactgatggtaagaggaatggagtgtctccttagagtgtgtctttctctgctctgtgtacttgactgcaccattagtggagtgctgcctgggcaggggtgtggccgtgctaacacacaagtcccaagtccttagagcaagttctgatacaccacatacccagccctggcctgtggGGACATGACTAGTGAGTGAGGCCCGCAGAGTAGACAGTCCTCAGATCTGTCATGTGATGCCAACAGGAACTGTGCTGGAAACGGGGAAGTAGGACCAGAAAGTCACATCTTCCAAACAAGTGCCTTGAGTTCACCATGGCGGCCTGGGGACACTTTTCCAGACCCACAGGCAAAACTCCTTGACTAAATTTGCTGGCACAGAGCACTGGGAATGTGGCTCAGGTGGTGTAGTGCTGACCTGGCATTGCTCAGTTTCTGGATCCAGTCCC

This mutation is a 390 bp deletion beginning at Chromosome 14 negative strand position 31,483,013 bp and ending after 31,482,624 bp (GRCm38/mm10). In addition there is a 9 bp insertion at the 390 bp deletion site (AGTGAGTGA) and 4 bp deletion (TAGT) 37 bp before the 390 bp deletion.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37965 TAA CAC ACA AGT CCC AAG TCC Wild type Forward A
37966 TCT ACT CTG CGG GCC TCA CT Common A
37967 ATG TAC TCC ACA CCT GAC ATG G Mutant Forward A
37968 Fluorophore-1 TAG AGC AAG TTC TGA TAC ACC ACA TAC Quencher-1 WT Probe
37969 Fluorophore-2 AAG AGC AAC CAT TCA AAG GGT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37965 0.40 uM
37966 0.40 uM
37967 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.