Protocol 34351: Probe Assay - Dync1li2<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

>chr8:104430159-104430251 93bp TCATTTCATGCCGCTGCT GCCTTAAGCAAACTGCTCTCAC

Mut= 90 bp

Wt= 93 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GCCACAGCACTATTCTTAAAACCAGAACACTGAAAGCCTGTTTAGGTTGGATGAAGCTGAAGGTGTCATGTGCCCATGCAGCTCACAGCTCAGTAGACAGAAGAGCTGGTGTTTGCATCATAGTCCCTAAACCCTAAAGCTGCAACCTCtgccatgcatagatgcttttgtttatatttattattacctttttatccccttaatttgctaagcctaagatgtgtttctatttgcgtctgtataatttttttcttttccctctgtagttatgaaagagtttcaagattatatcgagcctgaagaaggttgtcagggttccccacagagaagagggcctctgacctcaggctcagacgaagacagtgttgccctccctctgggtgacaacgtgctgactcataacctggggatcccagtgttggtggtgtgcacaaaggtacgtccagggaattgtgagcttggctgggtttgcccagtgaacaagaaagagttttagaagcttcgatggagtttcttctcaagggaaaattgggcttattcaggattcatcaaggtatagtgaatgtgtcagggtaggacattctgtcatttcatgccgctgctgaattgacagtacctagttcagaaagccctgttgacTTCATGGGCAAACTCAAGTGAGAGCAGTTTGCTTAAGGCTCTAGTTTATCTGGTCCAGACTGGTGGTGACATAGAATAATGAAGTAGAAATAGAAATTAAGCTTCATTTTAGAGAAAGTATGTCTCTTATAAGAGGGGGAAGGTAGCTTTAAAAACATCCTGGATTTC

This mutation is a 490 bp deletion beginning at Chromosome 8 position 104,430,198 bp and ending after 104,430,687 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
43381 TCA TTT CAT GCC GCT GCT Wild type Forward A
43382 GCC TTA AGC AAA CTG CTC TCA C Common A
43383 AGA AGA GCT GGT GTT TGC ATC Mutant Forward A
43384 Fluorophore-1 CAG TAC CTA GTT CAG AAA GCC CTG Quencher-1 WT Probe
43385 Fluorophore-2 AAG CTG CAA CCT CTT CAT GG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
43381 0.40 uM
43382 0.40 uM
43383 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.