Protocol 34816: Probe Assay - Ssu72<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

>chr4:155731509+155731619 111bp CACTAGGCTGGTAGACTTATGCTG GCTTGCTTCTCACAGGTTGTC

Mut= 98 bp

Wt= 111 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GACCATCCGTCGTCTAAGGTCTACTCTGTGCTGCTTTGGACACTTAGAAACAAGCATCTGTGTGGCCTGATCTGTCTTGAGAGGAGACAGTCCCTGCCATGCCTCTACTTTCCAGTTACAATGGCAAGGCTTTCTGGAGCCATTCTGTGGGTTTGGGTTATCGTGGGAGTTATGATCTctctacgaagtcagcaccttccctgtgcttcccatgttagggcagtggtgaccctgcttgtgttcaggcctagagctatgttgcccctgttgacaatggccaacttggaaccccctagaggcaaggggccattgacaggtctaggttagtaactatctttaatccaaagctccttgactctggtgttcagctatacacaaaatggaattttacatatgttggacagaaataaaaggatcaagccccgtccagaaaggttccagaactgcacggacctgtttgacttgatcctcacttgtgaagagagagtctatgaccaggttgtagaaggtgagtgcacagagcctgacctgctgttcaggttgtaccaccaacactaggctggtagacttatgctgccctgggcgacagtgatggagacagagggtgcccccacatcCTCTGACCTGTATGGACATCTGGAGGACAACCTGTGAGAAGCAAGCAGAAGACTGATGTCCAGCAAAACTCAAGCCTTGTGGCCTTCAATGTTTGCTTTTCCTATGTTAGGCCTGACATTTTATAGAACATGCTATTGGTGCCAATACTGACTGGCATAGGATGGCTTGAGGATTCTATCTAGGATTCTTA

This mutation is a 439 bp deletion beginning at Chromosome 4 position 155,731,135 bp and ending after 155,731,573 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44545 CAC TAG GCT GGT AGA CTT ATG CTG Wild type Forward A
44546 GCT TGC TTC TCA CAG GTT GTC Common A
44547 AGG CTT TCT GGA GCC ATT CT Mutant Forward A
44548 Fluorophore-1 CGA CAG TGA TGG AGA CAG AGG Quencher-1 WT Probe
44549 Fluorophore-2 CGT GGG AGT TAT GAT CTC TCT GA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44545 0.40 uM
44546 0.40 uM
44547 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.