Protocol 18274: End Point Analysis Assay - Ghrhr<lit>

B6;C-Ghrhr^lit Prkdc^scid/BmJ

Stock No: 010816

Version 3.0

Notes

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

WT - A/A

Het - A/G

Mut- G/G

 

 

Sequence

tcagaatctcctccctcataggttaccccatgtctcctccttctccacctagGATGCCCTGGGACCTGGGa/gTGGGCTGCTGTGCTGGCCCCCGACAGGCTCTGGCCAGTGGGTCTCTCTCCCCTGCCCTGAATTCTTCTCTCACTTCGGCTCAGACACAGgt

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction	Note
17752		TCA GAA TCT CCT CCC TCA TAG GT		Forward	A
17753		CTG TGT CTG AGC CGA AGT GA		Reverse	A
17754	Fluorophore-1	GAC CTG GGA TGG GCT GC	Quencher-1	WT Probe	A
17755	Fluorophore-2	GAC CTG GGG TGG GCT GC	Quencher-2	MUT Probe	A

Reaction A

Component	Final Concentration
Kapa Probe Fast QPCR	1.00 X
ddH2O
17752	0.40 uM
17753	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	--	repeat steps 2-3 for 40 cycles

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
010816	B6;C-Ghrhr^lit Prkdc^scid/BmJ
005764	C3.B6-Ghrhr^lit/J
000533	C57BL/6J-Ghrhr^lit/J
3 strains use this protocol