Protocol 37089: Probe Assay - Ehbp1<em1(IMPC)J>Alternate1
Version 2.0

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

 

>chr11:22172724-22172872 149bp TCTCCAGTTCTCACTGTCCTG GCATTCATCCTCTGAAAACA

 

Mut= 147 bp

Wt= 149 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

 TTATAGGTTTAGCAAGAAAACACCATTTGATTACATTAATTTAATCAAAGAGAGAAAAAAAGCAGGTAAGGGCACTTACAGTGCCCTTCTTGCCTATGATAGATCTTTTTCTCACACTAGTCTTGGAGCCATTCCCAGTTTTCCTGAGGCAGGGTCTTACATAGTTGAGGCTAGCTGTGAATTCTGGATCCTTGTGCCTGTATCTACCTTAGTGCTGGACTTATAGGTGTGAGCTCTACGTGTGCTCGTCACTAGTTTCTGTTTCACATTCTCCTCTagtataaatatttatgcctgggccatctctttctgttttacttagatataaaagctgtaataaaagctcatcacaattttattattgcattacaagaatttttttagcaaaggaagttcttgtttcttttttattggctataaaataattccaaactacataattatttttttgtcttccaggaatccccttctggtcggaggaaagctcttgccaccagcagcattaacatgaaacagtacgcaagccccatgccaacacaaactgacgtcaagttaaaattcaagccgctgtctaagaaggtcgtgtctgccactctccagttctcactgtcctgtatcttccttcgggagggaaaagcaacgtaagtgactcccttCCCGTcttagtGGATGGGCGCAGGGCAGACATCCATGCACGCCAGCCCTTCTCCAGCTCTCTGCTTTGTTTTCAGAGGATGAATGCCCTTGTTATTTTAAAATCCTGTCTTGATTCTCCAGAGCAACAATGACATAAAAAGTGTAACTGTTACCATTTCAAAATCAGTATGAGTTGACCTAGTGATGTGCACCCCAGTCCCAGCACTCAAGGCAAAAACATTTGGTACCACTGGCTGCCATTTTTGTC

This mutation is a 379 bp deletion beginning at Chromosome 11 position 22,172,810 bp and ending after 22,173,188 bp (GRCm38/mm10). In addition, there is a 6 bp deletion CTTAGT 4 bp after the 379 bp deletion.  

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48979 GCA TTC ATC CTC TGA AAA CA Common A
50477 TCT CCA GTT CTC ACT GTC CTG Wild type Forward A
50478 AGT GCT GGA CTT ATA GGT GTG AG Mutant Forward A
50479 Fluorophore-1 CCT TCG GGA GGG AAA AGC Quencher-1 WT Probe
50480 Fluorophore-2 ATT CTC CTC TCC CGT GGA TGG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48979 0.40 uM
50477 0.40 uM
50478 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.