Protocol 34145: Probe Assay - Asap3<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

>chr4:136229606+136229692 87bp AGGGAGCTGTTGTGAAAGGA GCTGCAATACCCTTGTCCAC

Mut= 104 bp

Wt= 87 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 ACTGCACCTGACTGGTTCTTGGCAACCCTGGGGAGAAGCTGGAGTGGGCTGAGCTTTGGGAAGGGAGTTGGGTAGGTGTGTTAGTGTCAGCTGGACAACAAGACTCAAGAGTGAGGTTCCCCCTGAAGGCAGCAGAGTGTCCTGTTTATCCCAGGGCCCctgctaccctttgggcaactcattcttttctcaactccaggattccaaaaagcatctggagaaggcgtggaaagactacgaaagcaaagtgtgagtgaccttggacgggtggccttcaggagagcacgtctgctctacattgaagaaggaggctgattttgggatgtgtcacttggggtgtctggctctttggagcctattgtcaggcctggctgggttctgggggccatgttgcaagatgaccaaagggagctgttgtgaaaggaccaggggatgctgcccCCTGGCCACCTTCTGGCCTTGTGATGTCCATGTGGACAAGGGTATTGCAGCCTCAAGATAACACAGCCCTGGGGCTGGAGAGATGGCTCAGTGGTTAAGAGCACCAACTGCTCTTCTGAAGGTCCTGAGTTCAAATCCC

This mutation is a 282 bp deletion beginning at Chromosome 4 position 136,229,360 bp and ending after 136,229,641 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
42909 AGG GAG CTG TTG TGA AAG GA Wild type Forward A
42910 GCT GCA ATA CCC TTG TCC AC Common A
42911 AAG AGT GAG GTT CCC CCT GA Mutant Forward A
42912 Fluorophore-1 CCA GGG GAT GCT GCC Quencher-1 WT Probe
42913 Fluorophore-2 TTT ATC CCA GGG CCC C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
42909 0.40 uM
42910 0.40 uM
42911 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.