Protocol 34421: Probe Assay - Cpsf4l<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

>chr11:113703640-113703758 119bp CCTTGTTTGGCAGTTGCAG TGCGTTAGGGGTGAGCTTG

Mut= 120 bp

Wt= 119 bp

Fam=Mut

Hex=Wt

Sequence

Mut Sequence:

ccttgtttggcagttgcaggcggcccgtgcctccaaggctgcctccttgtagaaatgcagctgaccaaggtccacagggcctgattgggggtcggtggggagggtacatgcctgcaatcc

This mutation is a 3917 bp deletion beginning at Chromosome 11 position 113,699,772 bp and ending after 113,703,688 bp (GRCm38/mm10). In addition, there is a 2 bp (TC) insertion at the deletion site.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
43571 CCT TGT TTG GCA GTT GCA G Common A
43572 TGC GTT AGG GGT GAG CTT G Wild type Reverse A
43573 GGA TTG CAG GCA TGT ACC Mutant Reverse A
43574 Fluorophore-1 TCA CCG CAT AGA CAC AGC C Quencher-1 WT Probe
43575 Fluorophore-2 AGG TCC ACA GGG CCT GA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
43571 0.40 uM
43572 0.40 uM
43573 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.