Protocol 21155: Probe Assay - Trp53<tm1Brn> Probe

STOCK Kras^tm4Tyj Trp53^tm1Brn Tg(Pdx1-cre/Esr1*)#Dam/J

Stock No: 032429

Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.

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=  145 bp

Wild Type = 63 bp

Sequence

Wt Sequence:tccatggattgctgtattggaatcaaacagaaatctatgtcattcacagcagtaacctcctgggaatacttcaagagacggagaaagggcgactgactgtgccctccgtcctttttcgcaatcctttattCTGTTCGAAtcataatttgt

tttcctgagacagggtttctctgtgtagtcctggctgtcctgggactcccctctgtagaccaggctggcctccaactgtctctgcctcctatgtgctgggatcaaaggtgcctgccaccaccgcctggcttcttagagttgg

 

Mutant Sequence:GAGACGGAGAAAGGGCGACTGACTGTGCCCTCCGTCCTTTTTCGCAATCCTTTATTCTGTTCGAtaagcttgatatcgaattcataacttcgtataatgtatgctatacgaagttatctgcagcccg

ggggatctgatatcaTCGAATCATAATTTGTTTTCCTGAGACAGGGTTTCTCTGTGTAGTCCTGGCTGTCCTGGGACTCCCCTCTGTAGACCAG

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction	Note
31852		CGT CCT TTT TCG CAA TCC T		Forward	A
31853		GAA ACC CTG TCT CAG GAA AAC		Reverse	A
31854	Fluorophore-1	CAA TCC TTT ATT CTG TTC GAA TCA T	Quencher-1	WT Probe
31855	Fluorophore-2	TGC TAT ACG AAG TTA TCT GCA GCC	Quencher-2	MUT Probe

Reaction A

Component	Final Concentration
Kapa Probe Fast QPCR	1.00 X
ddH2O
31852	0.40 uM
31853	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
026512	B6(Cg)-Tg(TPO-cre/ERT2)1139Tyj/J
032435	B6.129-Kras^tm4Tyj Trp53^tm1Brn/J
037954	B6.Cg-Gt(ROSA)26Sor^tm4(CAG-cas9*/GAG-POL,-mNeonGreen)Tyj Trp53^tm1Brn/TyjJ
033754	STOCK Brca2^tm1Brn Gt(ROSA)26Sor^tm3(CAG-EYFP)Hze Trp53^tm1Brn Nkx3-1^tm4(cre/ERT2)Mms Pten^tm1Hwu/AbshnJ
033755	STOCK Gt(ROSA)26Sor^tm3(CAG-EYFP)Hze Trp53^tm1Brn Nkx3-1^tm4(cre/ERT2)Mms Pten^tm1Hwu/AbshnJ
033756	STOCK Gt(ROSA)26Sor^tm3(CAG-EYFP)Hze Trp53^tm1Brn/Trp53^tm3Tyj Nkx3-1^tm4(cre/ERT2)Mms Pten^tm1Hwu/AbshnJ
032429	STOCK Kras^tm4Tyj Trp53^tm1Brn Tg(Pdx1-cre/Esr1*)#Dam/J
7 strains use this protocol