Protocol 35263: Probe Assay - Gulp1<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

>chr1:44708500+44708641 142bp AAAGGTTCAGGAAACTAGACTG TCAGGCGTATGCATCCATGT

Mut= 140 bp

Wt= 142 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 CCTTTTAGATTATGCAAACCCTGTTGCCAAAGTTATCATAACTATAGAAGAACAAAATATTTTCTTATCTGTCAATTATTAAATATATCTTGGCATCTATAAAAGCAGTAAACACAAAATAAAAGTTGTCTTAATTTGCGTAACTGTTATTACTCATAGCTCCCTGCTGATCTTATGAGgtgaggaccaggcgaagtgatatatgtgtaaaagtacagcaagtttggccttagccttggctccatactggtattgcacaaaattaacagatgtattttgtggttaaaaaaaaaagaataaatgccttgatcatttccagttacaatatttctttctttaactaacaaaggttcaggaaactagactgtgctccccagcaagacttccttcaggtgagaatgtatgtatatgtcatcatagaaatttgtggtttaattcagtactgcattttctctgttacagacaaaacatggatgcatacgcctgaagctttatcaaaacattacattccctataatgcaaaggtaaaatggttattgagatattatctatttttacattgttatatgttatgttgaaataattgtgctaattaaaaaaaaaacttatgcattggcatgtcctgGTAAGCAATATTTGAAACTAATTTCATATTACTGAAATAAGTAATATAACATAAAAAATTGTTTTTATATCCAAATTTCTTTGAAAAGAAAGGGATACTCTGTTTAGATTCTCTATAGTCATGTATTTCACTGAAGCTTTAAAAAAAA

This mutation is a 446 bp deletion beginning at Chromosome 1 position 44,708,334 bp and ending after 44,708,779 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
45638 AAA GGT TCA GGA AAC TAG ACT G Wild type Forward A
45639 TCA GGC GTA TGC ATC CAT GT Wild type Reverse A
45640 TCA TAG CTC CCT GCT GAT CT Mutant Forward A
45641 AGA GAA TCT AAA CAG AGT ATC CCT TTC Mutant Reverse A
45642 Fluorophore-1 CAA GAC TTC CTT CAG GTG AGA AT Quencher-1 WT Probe
45643 Fluorophore-2 ATG AGG TAA GCA ATA TTT GAA ACT AAT TT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
45638 0.40 uM
45639 0.40 uM
45640 0.40 uM
45641 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.