Protocol 32354: Probe Assay - Tomm20<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

>chr8:126940894-126940992 99bp TGATTGGATTTTCCTTTCTTCC TGGTTCTTTTGCTACTGTGCTG

Mut= 90 bp

Wt= 99 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 

CTGGAATCTGTATATTTCAGTAATAATGGCTTAATTATTTTATTTAGAAAGGGGGCTTTGTTCTGCTGCTGCACAGCTGTTGGGACAGTCTCTGTCTTCTGAATTGTTGGACCTGGGGATGCAGAAGAAAAGAGAAAGATCCTACCTAGAAACACCAGTTCACTCGCCAGGGAGgcagggattctggaggcaggaacgttttgtagaattaatgatggttgcagcaattagatgtcttgatccttaatgatgatagccaagtttttaatttatttttcttgtaggaagaaagaaacagaagcttgctaaggagagagctgggctttccaaggtaatgaaaatagtcttgtgtttttgcgtgtagattgcatgctctctaagcctcctgtgttttcctttggtggtggtaagagcaaatatgttaattgggtaagcagcaagatgagagtctccagagatggacatgaagagttaaagttctcggctacttaaaaatggttttaaaaaatacagcatttgattggattttcctttcttccttccgacagtaacctttaatcctagtttgagAAGCTCTGTTAGAGGCAGCCTGGGCAGCACAGTAGCAAAAGAACCACGTGTGTTAGTCCCACAGATCTTTAAGGTTTTTACACCTATGGATCAGAATTATATCAGGTTCTGACATTTAAAAAAACAAATTGTAATTTGTAGAATATAAATTTTCATTTTTTTCTTTTCATAGGTCCTTAGTTTACAATTTTTAATGACTGTAGGTTTTTATCTACTTAATT

This mutation is a 396 bp deletion beginning at Chromosome 8 position 126,940,940 bp and ending after 126,941,335 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
38903 TGA TTG GAT TTT CCT TTC TTC C Wild type Forward A
38904 TGG TTC TTT TGC TAC TGT GCT G Common A
38905 AGA GAA AGA TCC TAC CTA GAA ACA CCA Mutant Forward A
38906 Fluorophore-1 TCC GAC AGT AAC CTT TAA TCC TAG TT Quencher-1 WT Probe
38907 Fluorophore-2 ACT CGC CAG GGA GAA GCT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
38903 0.40 uM
38904 0.40 uM
38905 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.