Protocol 34831: Probe Assay - Scarb1<tm1Okoch> Probe

B6;129S-Scarb1^tm1.1Okoch/J

Stock No: 032062

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

>chr5:125280934-125281033 100bp GCATCTTCCTGGATGTTATTCA CTGAAAGGGTCCAAGAAACC

Mutant=  110 bp

Wild Type = 100 bp

Sequence

Wt Sequence: gcatcttcctggatgttattcacatggagagcagttaccctcctgcctctcaccctcctgcgagatgggaatcttgCCtcggtttcttggaccctttcag

 

Mutant Sequence: TACGTTCGTGGGATTGTGTCCGTGTCGCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGTTGGTACCGTACGCGGACGACCAActcggtttcttggaccctttcag

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction	Note
44605		GCA TCT TCC TGG ATG TTA TTC A		Wild type Forward	A
44606		CTG AAA GGG TCC AAG AAA CC		Common	A
44607		TAC GTT CGT GGG ATT GTG TC		Mutant Forward	A
44608	Fluorophore-1	TTG GTA CCG TAC GCG GAC	Quencher-1	MUT Probe
44609	Fluorophore-2	CAG TTA CCC TCC TGC CTC TC	Quencher-2	WT Probe

Reaction A

Component	Final Concentration
Kapa Probe Fast QPCR	1.00 X
ddH2O
44605	0.40 uM
44606	0.40 uM
44607	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
032062	B6;129S-Scarb1^tm1.1Okoch/J
033709	STOCK Scarb1^tm1.1Okoch Ldlr^tm1Her/J
2 strains use this protocol