Protocol 31718: Probe Assay - Rbm6<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

>chr9:107847034-107847158 125bp TCCAGAAGTTTCCTGTTGGTG CAGGCACTCAGAAGAACTGGA

Mutant=  92 bp

Wild Type = 125 bp

Sequence

Wt Sequence: GAACCTCATTCTTAACCCAGCTCTTATTCCTATGTATATTGTATTCAGTTTCTGTTCCTTTCTCTCTGTGGTGTGCTTCCCTCTGTTCATGGTGACATAAAGCCCGtgtgattcttggccagctgggattataggtttgtcattatgcctaatttcatttagcacattttgaattcttacagtgtatatggtttaattttgtcttcataaaaatttgtagatttagttatataaagtaaaaaccagtagggaaataaaaaattagcacttaacatgctttgtataattagtgtaaatgtcatatttttgcagattcttaatgcctttcgaacttctgatggcatacctgtaaaggacttgcagttgaaggagtataacacaggtgagttttttagatgtgcatagactcttggtttaggaagggtcttggagatatgtctgcatcatatgctactcaaaaatttgttttttaagaaatcacaagtatgatttccagaagtttcctgttggtgccttcaaataacaagcagccttagtcttaactgtgattctttgtatggctctttgtccacacATGGGCGATGAGGATGCGTGTTCCAGTTCTTCTGAGTGCCTGTGAGATATAAAGTGAGTGTCATAGCATCTATCCCTGTTTTATATGATTAAATTATTAAACCTAGTATTTATGTTCTTCACAATGGTGGTATGTTTACATTTGAGCCTA

 

Deleted Sequence: tgtgattcttggccagctgggattataggtttgtcattatgcctaatttcatttagcacattttgaattcttacagtgtatatggtttaattttgtcttcataaaaatttgtagatttagttatataaagtaaaaaccagtagggaaataaaaaattagcacttaacatgctttgtataattagtgtaaatgtcatatttttgcagattcttaatgcctttcgaacttctgatggcatacctgtaaaggacttgcagttgaaggagtataacacaggtgagttttttagatgtgcatagactcttggtttaggaagggtcttggagatatgtctgcatcatatgctactcaaaaatttgttttttaagaaatcacaagtatgatttccagaagtttcctgttggtgccttcaaataacaagcagccttagtcttaactgtgattctttgtatggctctttgtccacac

468 bp deletion beginning at Chromosome 9 negative strand position 107,847,543 bp and ending after 107,847,076 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37478 TCC AGA AGT TTC CTG TTG GTG Wild type Forward A
37479 CAG GCA CTC AGA AGA ACT GGA Common A
37480 CCT TTC TCT CTG TGG TGT GC Mutant Forward A
37481 Fluorophore-1 CAA GCA GCC TTA GTC TTA ACT GTG Quencher-1 WT Probe
37482 Fluorophore-2 TCT GTT CAT GGT GAC ATA AAG CC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37478 0.40 uM
37479 0.40 uM
37480 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.