Protocol 35671: Probe Assay - Bag2<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

>chr1:33747215-33747334 120bp TTCTGATGAATATGCAGTGTCC TGTGTCCACAGAAGGCAGAG

Mutant=  134 bp

Wild Type = 120 bp

Sequence

Wt Sequence (deletions in lower case):

GGTGGTATATTCTGATGAATATGCAGTGTCCCCGCCCCCGCCGTGAGCCCTCCTCTGGCTAACctctgctcgctctgaagttgcttggctgcatcccagctgcccatgtctctgccttctgtggacacatagactcaggatgatctctggccctaactgtgtgaagacttttcctcagttatgctgatatgccgtagtacttttggcttttacctttagaaaaatttacagaagtgcttacagaaactaaagatagcgcaagtgcatgctgtggcgttgctgacgtgggaagtttctgcctcatactgactgatgtttcgtgtttaggagaaagagaggaactaaacctgactgccaaccgtctgatgggccggaccctcacggttgaggtctcggtggaaacgatccgaaacccccagcaggaggaatccttgaagcatgccacgaggattatagacgaggtggtcagcaagttcctagatgacctggggaatgccaagagccacttaatgtcactttacagtgcctgctcatcggaggtgccgcctgggccggtggaccagaagtttcaatcgatagtcatcggttgcgctcttgaggatcagaagaaaatcaagaggcgattggagactctgctgaggaacattgacaactccgacaaggccattaaactcctagagcatgctaaaggagctggttccaaaagcctgcagaacactgacggcaaatttaattagccttcaaattcaccggcccttacaaaagatgtagaaaagaaaactactattttcaatagctaggtctctatgttaggcataaccacttaattgatattggtaattatttcagatggggaagtattccagtgttggcagacacaggtaacacttaacactaggaatatttcagtcacctactttgaagttttggacagtgttcccatcttgaactggactctagcactgtatatttaacttctgaagatgcacatgtagtgtgtgtgtgtgtggggggagtaaaaaaagagggggagcagcaaaagtcccactgccctttccttgacttcagcacagagcatccagcctctggagcagtgcactcacatcgcagtgtgagcagagcacccagccctggagtggtgcattcacaccacagtgaacagaacacccagccctggagcagtgcactcacaccgcagtgtgaacagaacacccagcccctggaacagtgtactcacagcacagtttgaacagagcacccagcccctagagcagtgtattcacacctcggtgtgaagagaacacccagcccccggagaggtgcactcacaccagtgtgaacagatcactcagcccttgagcagtgcactcacagcacacagtgtacctagaaggataagcatatcagaacttcaccctgcatggtctccatgtttcaggcatctgttttctctatgcatatgcatctcataccatgaatttttaggtgtgcgtggaagcatgacaaatgtgaggagcccaccttcattttaccactgtgaagctccgcccccaccccagggccagtgctgaccgctctcttcatgtgctgccttgagctcacgcgtgtgcggcgggagcacttgcctaaccacttccctgtgggcagttggatggtttcaatttgttgctgtgactacaacatacttgtatctgctgagctttattctgttcttggtcttttttatataaaattccattaatgggttcatcaggccaaaggatatatacattttcatggtctagaaacatatttctaaatttttataagttatatcagtttgtactgtcttcagtaattaaaagaatccatttcgctgaacttctagcattggccattatttttctaccgtcagacacaaagccttctatttaatgggtaccttgtttcctaagaaggctggccagcgtggtcatcttCCCGAGTTCTTTCTTTGTTTTTTGTTTTTCGAGACAGGGTTTCTCTGTATAGCCCGGGCTATCCTGGAA

 

This is a 1895 bp deletion beginning at Chromosome 1 position 33,745,386 bp and ending after 33,747,280 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46693 TTC TGA TGA ATA TGC AGT GTC C Common A
46694 TGT GTC CAC AGA AGG CAG AG Wild type Reverse A
46695 TAC AGA GTG AGT TCC AGG ATA GC Mutant Reverse A
46696 Fluorophore-1 CTC GCT CTG AAG TTG CTT GG Quencher-1 WT Probe
46697 Fluorophore-2 CCT CTG GCT AAC CCC GAG T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46693 0.40 uM
46694 0.40 uM
46695 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.