Protocol 35469: Probe Assay - Klf16<em1(IMPC)J>-Alternate 2
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

>chr10:80576732-80576828 97bp AGCCACCGCTGTCCCTTC ATACGAGGGGCTTACCTGTG

Mut= 80 bp

Wt= 97 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case) point mutation in brackets with wt first:

 CGGGGCGGGACTGAGCGCGGGCGGCGGCGGCGCGCCGGGGCCTCCTCCTCCCTTCCCTCCTCCGCCTCCCGCACTCTAGCAGCCGCTGCCGGCCGGACGGGCGTAGGTCCCACCGCCTCCGCTTCTCTCCCGCCGCCGATTCCGTGTCTCCG(C/A)GTCCTTGcccggcatgtcggcggccgtggcgtgtgtggattactttgccgccgacgtgctcatggccatctcctcgggagccgtggtgcatcgcggacgacccggccccgagggcgcgggccccgctgccggtctggatgtgcgcgccacgcgccgcgaggccacgccgccggggacccctggggcaccgccaccgccagctacggctccggggccgggaggcgccaccgctgcgccccacctgctggccgccagcatcctggccgatctgagaggcgggccggtagtcgccaccgcagccagcaccgcggggggcacctctccggtctcctcctcctcagccgcttcgtctccctcttcgggccgcgcccccggcgccgccaaaagccaccgctgtcccttccatggctgcgccaaagcctattacaagtcttcgcacctcaagtcacacCTGCGGACTCACACAGGTAAGCCCCTCGTATTTTACACAGGTGGGATGCAGGGAGTGCATTAGAGAGGTCTCGGGGACCCCGTTCCCCCATCTCACTTGGAACCCCGAAACTGCGTGGCGCGTGGGGGGCGGGGAGGGGGGCGCGCCCAGGC

This mutation is a 444 bp deletion beginning at Chromosome 10 position 80,576,763 bp and ending after 80,577,206 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
42662 AGC CAC CGC TGT CCC TTC Wild type Forward A
42663 ATA CGA GGG GCT TAC CTG TG Common A
42664 ACC GCC TCC GCT TCT CTC Mutant Forward A
43625 Fluorophore-1 CGC CAA AGC CTA TTA CAA Quencher-1 WT Probe
46159 Fluorophore-2 CCG AGT CCT TGC TGC GGA CT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
42662 0.50 uM
42663 0.50 uM
42664 0.50 uM
Glycerol 6.50 %
Dye 1.00 X
Kapa 2G HS taq polym 0.03 U/ul
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.