Protocol 33341: Probe Assay - Utp20<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

>chr10:88822194-88822288 95bp ACAGCCAAAGCAGCAAGACT TTGAGAAGAGGGGAGAACCA

Mut= 81 bp

Wt= 95 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

AATGCAGAACTGAAGAGTCCTACATTTTTTCTCTGCAGGCAGTTTTTCCTGTGACTATTACTTGGGCTTTAAACAGTTATTTGCTACAGCCAAAGCAGCAAGACTTGTTTTTGTAATTCACCTGctgctggtagacctgctgctggtagactgaggaagttggttctcccctcttctcaaaaacacaaaaagggggtttttaaccccttatccttgtcctaagatgctggctgtggtccgtgctctgcagcttggtcagttgtcctggagttgcctcactaacagctgccttgctcttggcaggacgtcgagacttacttcttcgaggctctgctgaagtggagagagctgaaccttacggaacatttcggtaccgtctgcttctttctacttcatcttcccataatttgcacatcttatgttcccaacaactagtgaggcgatggagaaatagaacataatcctttttgctttctttcttttttccttttgttaatccagggaaattttacaaagaagtcattgacaaatgtcagtcttttaaccagttggtctatcatcaaaatgaaattgtccagagcttgaagactcacctgcaaattaggaacagtttggcctaccaacctcttttggagtaagtcacatctcagatgaaggctcaatagtgtttatctgagttctaaggaaaaccatgcttcatcactggccttaaattacggtctgtaatttagaagcaggatgagtatgccttccctgttagtttactgtatcagaaaaagatgtgtttctgtaaagcagcatcctggtgtacattgCACTTTCTCTGTGCTCCTAAGAGAGCTGTATGTTTCCTTGAAACCAGGGATGGGTGGCCCCAGTTCTGCTATCACTGTTTCTGACTTGAGACCAGGGATGGGTGG

This mutation is a 691 bp deletion beginning at Chromosome 10 position 88,821,559 bp and ending after 88,822,249 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
41179 ACA GCC AAA GCA GCA AGA CT Common A
41180 TTG AGA AGA GGG GAG AAC CA Wild type Reverse A
41181 TTC AAG GAA ACA TAC AGC TCT CTT AGG Mutant Reverse A
41182 Fluorophore-1 CTG CTG CTG GTA GAC TGA GGA Quencher-1 WT Probe
41183 Fluorophore-2 CAC CTG CAC TTT CTC TGT GC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
41179 0.40 uM
41180 0.40 uM
41181 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.