Protocol 32609: Probe Assay - Arhgap21<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

>chr2:20890030-20890127 98bp TTATTTGGTCTGGGTGTGGA TGTCGAAACTCCCAACACTG

Mut= 89 bp

Wt= 98 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 

GTGTCTAGAATTCTCAGAACATTGCTATATTTCATGAGCAGTTTGATGTTTTAAAAGGAAGATTTATTTTTGTAACAGGTACTTTAAAAATGTGCAGGCATATTTTAAAAAATACGGGCTTATTTGGTCTGGGTGTGGAGCTCAGACAGCaacgggtttgatcccagcaccatggaaacagagtgaagaggagggagcagtgttgggagtttcgacacagttactcagagtgagtagttctttcaaggagttcctaatatgttgttctccccccccccccctcttctttgaaggtgacagaattataaaagtcaatggagaaagtgtcataggaaagacctattcccaagtaattgctctcattcagaacaggtaagagtcttgaatcctgattaatatgggctaaaaccatatagcacaggtagcttttatacagttctgtgctcctaggacaagggtaacacagaatttcctccaaaactgcaagttgtgtatggtattgccaaatgttagcccactgattgttgagtgggtttgcaagttgtaatattcattgtaatgtttacaatgcaaaatcctatgGGTTCTGAGAGCACTCATTATAGATTTAAAATATGAGTTTTCCACTTTGATTTCAAGTGATAGGTATATAATTTGTTTAAATATAGAAATAAATGCACACTTGGTTTTCTAAAACAAGTCTTACTGACATTCATATTT

This mutation is a 432 bp deletion beginning at Chromosome 2 position 20,889,665 bp and ending after 20,890,096 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
39549 TTA TTT GGT CTG GGT GTG GA Common A
39550 TGT CGA AAC TCC CAA CAC TG Wild type Reverse A
39551 ACT TGA AAT CAA AGT GGA AAA CTC Mutant Reverse A
39552 Fluorophore-1 TGA TCC CAG CAC CAT GG Quencher-1 WT Probe
39553 Fluorophore-2 ACA GCG GTT CTG AGA GCA CT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
39549 0.40 uM
39550 0.40 uM
39551 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.