Protocol 36558: Probe Assay - Notch2<tm3Grid> Probe

STOCK Notch1^tm2Rko Notch2^tm3Grid Gt(ROSA)26Sor^{tm9(CAG-tdTomato)Hze} Tg(KRT5-cre/ERT2)2Ipc/JeswJ

Stock No: 030855

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

Mutant=  85 bp

Wild Type = 100 bp

>chr3:98071488+98071587 100bp CCTGGGAGGGATGTTTCTCA AGCCTTTTCCCCATATTCCAAC

Sequence

Wt Sequence: GGTCAGGTTTCAACCCCAGATAGGAAGCAGCTCAGCTCACAGCCTGCCCTTGTCTCTGGGCTTCCTGCAGTAGGGGCTCCCCAGGCAGCAGTGCCAGCTGTTAGATACCAGCCTGGGAGGGATGTTTCTCATTCAAACTcccacacccgggtccCCCAACTCCAGTGCACGTTTAGCGTTATAATACAAGTTGGAATATGGGGAAAAGGCTCAGGGACACTTAAAAAAATCTGTTAATAGATAAATGTTAGCTATTTTAAACAAGTA

 

Mutant Sequence: GGAAGCAGCTCAGCTCACAGCCTGCCCTTGTCTCTGGGCTTCCTGCAGTAGGGGCTCCCCAGGCAGCAGTGCCAGCTGTTAGATACCAGCCTGGGAGGGATGTTTCTCATTCAAACTCCCACACCCtgcagataacttcgtataatgtatgctatacgaagttatCCCGGGTCCCCCAACTCCAGTGCACGTTTAGCGTTATAATACAAGTTGGAATATGG

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction	Note
49090		CCT GGG AGG GAT GTT TCT CA		Common	A
49091		GGA CCC GGG ATA ACT TCG T		Mutant Reverse	A	Mut R
49092		AGC CTT TTC CCC ATA TTC CAA C		Wild type Reverse	A	Wt R
49093	Fluorophore-1	CCC ACA CCC TGC AGA TAA CTT C	Quencher-1	MUT Probe
49094	Fluorophore-2	CTC CCA CAC CCG GGT CC	Quencher-2	WT Probe

Reaction A

Component	Final Concentration
Kapa Probe Fast QPCR	1.00 X
ddH2O
49090	0.40 uM
49091	0.40 uM
49092	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

Stock Number	Strain Name
010525	B6.129S-Notch2^tm3Grid/J
030855	STOCK Notch1^tm2Rko Notch2^tm3Grid Gt(ROSA)26Sor^tm9(CAG-tdTomato)Hze Tg(KRT5-cre/ERT2)2Ipc/JeswJ
2 strains use this protocol