Protocol 36501: Probe Assay - Ccnt1<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

>chr15:98554425-98554525 101bp ATGGGAGAAAATCTTGTGAAGG AAGTGACCAGGGTTAATGATCTG

Mut = 99 bp

Wt = 101 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

 CTACATGAGTGTAGGGCACCACCTGCATACGTGCCTGGGAAGGCCAGGGAGTGAGTGTTGGATTCCCAGGAACTGGAGTTACAGATGGCTGTGAGCTTTCTGTGAGTTCTTGAAAGCAAACCTAGGTCTTCTGCAACAGCAGCAAGTGTTCTTAACTGCAGAGCCATTGCTCAGGGGCCTCTATCCAGACTTTTGATTGTTTTGTTTTTAAAGTTATTTACATCCTTTTGctggatgtaacttctgggttttttttccccttcttaggcttacctacaacaagttcaagatctggtgattttagagagtataattttgcagaccttaggtaagttaattttcctttttaaagcaggaaaaaaatatgtatgggatataatgatttcatgttaaagtttgttagatgggagaaaatcttgtgaaggctttgggaagtgtctgttcctgggtaaaaatgtctgTGGGGCTCATTAAATGGAGACAGATCATTAACCCTGGTCACTTTTCACAGTGTTAGGTCCTTGGTGACCCTTGAAACAGCTATTAGTCAAGATTTAAAAAAATTTTCTCATTGTAAGGTTTATGATTTTTTTAAAAAAAGTTTTATTATATATAAGTACACTGTAGCTGTCTTCAGACATCCCAGAAGAGGGTGTCAGATCTCATTACAGATGTTTGTGAGCCACCATGTGGTTGCTGGGAATTGAACTCAGTACCTCTGGAAGAGCAGTCGGTGCTCCTTAATTTTTATTTATTTATTTATTTT

This mutation is a 231 bp deletion beginning at Chromosome 15 position 98,554,468 bp and ending after 98,554,698 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48929 AAG TGA CCA GGG TTA ATG ATC TG Common A
48930 ATG GGA GAA AAT CTT GTG AAG G Wild type Forward A Wt F
48931 GGG CCT CTA TCC AGA CTT TTG Mutant Reverse A Mut R
48932 Fluorophore-1 AGT GTC TGT TCC TGG GTA AAA ATG Quencher-1 WT Probe
48933 Fluorophore-2 ATC CTT TTG TGG GGC TCA TT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48929 0.40 uM
48930 0.40 uM
48931 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.