Protocol 33754: Probe Assay - Alg3<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

>chr16:20608666-20608782 117bp CCTTGGTGCTTGTTTTCTTGA GTCAGCTCCCCAAACCTGA

Mut= 110 bp

Wt= 117 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 CACGGCACTGGAGAGGCAGGACAAGGGATCCTTGAAGCAAACTGACTAGCTAGACTCTGTTATATAAATGACAGAGGAAGACACCAGTGTCAACTCCTGACCTTCATATGCATGCACACATAACACACAGACGAAATATATGGGTTCACCCAGAGaatgccgtaggtgtggttacatcagggacgataatgggagaaacctgggaatgaggtagaatctctgggctttggtggtctgaaggcatggggtttagaagtaggcaagttctggactcagaaggaacgagaccatgttatcttcttccagacacagagattgactggaaggcctacatggcccaggtggagggtttcatcaatggcacatatgactacacacagctgcagggtgacaccggacctcttgtgtgagtgaggataggaagtctgaaggcattgtgaagggctgggtgggtaccaggctgatagtcctgccctaggctcatatttcctttcatcttccagctacccagctgggtttttgtacatctttacggggctattctatgctactgaccggggcactgatatccctatggcccagaacatctttgctgtactctaccttgtcaccttggtgcttgttttcttgatataccaccagacctccaaggtgagttgatgccacaggaccaactggccctggcctgaagTGGGTGGTGGGGGTGGCTCAGGTTTGGGGAGCTGACGTTGGTGCCCACCCTTTAGGTACCTCCCTTCGTCTTTTTCTTCATGTGTTGTGCCTCTTACCGTGTCCATTCCATCTTTGTACTGCGGCTCTTCAATGACCCAGTAGCTATGGCCTTGC

This mutation is a 536 bp deletion beginning at Chromosome 16 position 20,608,702 bp and ending after 20,609,237 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
42125 CCT TGG TGC TTG TTT TCT TGA Wild type Forward A
42126 GTC AGC TCC CCA AAC CTG A Common A
42127 CAC CAG TGT CAA CTC CTG ACC Mutant Forward A
42128 Fluorophore-1 ATA CCA CCA GAC CTC CAA GGT Quencher-1 WT Probe
42129 Fluorophore-2 CAC CCA GAG TGG GTG GTG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
42125 0.40 uM
42126 0.40 uM
42127 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.