Protocol 36408: Probe Assay - Tbc1d32<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

>chr10:56196823-56196927 105bp AAGTGTTTGGTCTGTGCTGCT GGTGAGAGGGTAAAAGTCTGTG

Mut= 107 bp

Wt= 105 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case; and insertion with carrots ^g^ (T insertion):

 TCAAAATGTAGTGTTGCCTACTGTGCCACTGTTGGATGGCACCATTAAAGCCTCCAGCACCTCTGTGCAAGCCTGCAGCTTCATTTCACAGCACCACGTGACCCTTCCTCCCGCCACCATTCACTCCTACTTCCTGCTGTGACCAGATTTCACAGCAGTGCATTTTGTTGTTGGTTTTACCCTTAGTTGTGGATAATTTTACTTTTGGCTAAATGAAGACATGCCCTTTAGAATAGACACGGCCTTCCCCTGG^tttctgcgt^AGTGTATCTTGTGTTTATTTTCTGTTCCATGCGAGAAGTGTTTGGTCTGTGCTGCTGTAAGATCGCAAATGAGGATGGGCTGTTttgcttccctaggaccggattttaaagttctacgcacagacttttaccctctcaccgttacatatgaccaaggagatttatacaagcttaggtatgttcaccaaatgacttcaattgttgttctcctatatgcacttaaaatcccgtaagaaatgacttcttccagaagcattgcgtaatatatataacgactgtatattttccctttccttttcagctaaatatctggaggtatattttctttctagagaaaatcaccttcctactctttcaactggagtagacataaccagtcccaatgtgacccgcttactgaaaaaggtatccacttactatgatgtactgtatggaaagtagggctatatttaagaattaaactcttttgagagcttttgtatactgcttataggttcgccttctgaatgagtaccagaaagaggctccatctttttggattcgtcacccagagaagtaagttccccaactcctgtgatgtaaggtataggctatgtccttcaattatttcaaggacagaaggaacagtagtcttgagcgatGGACATGGTTCTATATGCCACTTAGTTTTTGTCAGCTTGACACCAAATAGGACCACCTAGGAAATAGACCCTCATCTGAGGAACTGCCCTCATCACTTGCCTATGTGTGTGTTTTTGTAACTGTAAATTGATATAGGGGGCCTCCCAGGGGCAATACCTTCTGTAGGCACATGGCCCTGGGCTGTATAAGAAAAGTAGCAAGCTTGAGGAGGCAAGACAGCAAGCAACGCCTCAATGGTTTTTGCCCCAAATTCCTGTCTTGAGCTCCTGGTCTGGCTTCCCTT

This mutation is a 557 bp deletion beginning at Chromosome 10 position 56,196,322 bp and ending after 56,196,878 bp (GRCm38/mm10). In addition, there is a 1 bp (T) insertion and a 9 bp deletion (ACGCAGAAA) 84 bp after the deletion.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48684 AAG TGT TTG GTC TGT GCT GCT Common A
48685 GGT GAG AGG GTA AAA GTC TGT G Wild type Reverse A
48686 AGG TGG TCC TAT TTG GTG TCA Mutant Reverse A
48687 Fluorophore-1 CTT CCC TAG GAC CGG ATT TTA A Quencher-1 WT Probe
48688 Fluorophore-2 ATG GGC TGT TGG ACA TGG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48684 0.40 uM
48685 0.40 uM
48686 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.