Protocol 46664: Probe Assay - Cdkl1<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

>chr12:69760226-69760326 101bp GGATTTGTGGGCTGATGG CAGCTCAAGAAACCCAATCC

Mutant=  100 bp

Wild Type = 100 bp

Sequence

Wt Sequence: ggatttgtgggctgatggcctgtggatccagaggaggctgtgTctcccgggatttttcagtcccaaagcctgcccccgtctggattgggtttcttgagctg

Mut Sequence: ggatttgtgggctgatggcctgtggatccagaggaggctgtgTCgttttttgtttttggtttttgtttttaatttgtaaaatgtgaggaagaaggaaccc

A 396 bp deletion beginning at Chromosome 12 position 69,759,888 bp and ending after 69,760,283 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
68571 GGA TTT GTG GGC TGA TGG Common A
68572 GGG TTC CTT CTT CCT CAC ATT Mutant Reverse A
68573 CAG CTC AAG AAA CCC AAT CC Wild type Reverse A
68574 Fluorophore-1 CGG GAT TTT TCA GTC CCA AA Quencher-1 WT Probe
68575 Fluorophore-2 AGG AGG CTG TGT CGT TTT TTG TT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
68571 0.40 uM
68572 0.40 uM
68573 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.