Protocol 14297 - Gata4<tm1.1Sho>

Notes

This genotyping assay uses pyrosequencing technology and is run on the Biotage PSQ 96MA. The Jackson Laboratory is not posting the complete details of our pyrosequencing genotyping assays as the primers for pyrosequencing cannot be used for sequencing using more traditional methods. The wild type and mutant nucleotides and the flanking DNA sequence are provided below.

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 = G/G
Heterozygote = G/T
Wild type = T/T

Sequence

ACAGTTCATGATCAAATCATCACATGGCCATACTTTTGAAAAGGAAAATGAAGAAAAG
AGTCTGAGTAGGTTAGGTGGATACTATAAAGATGTCTAGAGGCAAGACATGAACTCAA
AAGGGTCCCCTAGAACCTGAGCAGCTGATGACTCTTATCTTAAACAGCCCTGGATGGC
AGAGAGTGGAACCCACTGGCCATTCGGGCCTAAGTGAGGAAGAATCAGTCTCCCGGG
AGACATCCTGCCCTGCTTTCTAGGTGAAAGGTCTCTGAGCAAAACAAACCATTTGTGT
CTTTTCTCCTGTGTCCTCCCCCAGTAGATATGTTTGATGACTTCTCAGAAGGCAGAGAG
TGTG(t/g)CAATTGTGGGGCCATGTCCACCCCACTCTGGAGGCGAGATGGGACGGGACA
CTACCTGTGCAATGCCTGTGGCCTCTATCACAAGATGAACGGCATCAACCGGCCCCTC
ATTAAGCCTCAGCGCCGCCTGGTAAGTGAGCATCTCTATAGCTTAGGTCCGCCAGCCT
GGAGAGCAGTCAGGTTCTCCCCTCCTCCCTGAACTTGGCTTTGAGGCTTGAAGGATA
TAAGCTGAGGGGTTAGGTATGTGGGAGGAAGGAAGGAGCCTCTCACACTCTTAGAG
TGAACTTGCACTCGCTCCAAAGATGCTGGCCCAGCTAATCTATCACAATGCAATAGA
GGAAAGTGAAGCTCAAAAAGGTGACTGGCAACAGACACTGGCCATGAGAAGCCAG
ACCCAGGTCTCCTGGACTCCAGGCCCTTTGTCTCGAAAGCCAACGTAAACCAGGATC
TGTCTTATTTTCAGTTGATCCCCTAGCACGACCCAGAGTCCAAGAAGGTAAAGCAGTA
GGTGTGAGTCCTGGTTGGGAAGGCCTAGAACTGGGGCCAGGTATTTGCCACCTGGAT
CTCTGCGCTCTCTCTCTCTCT

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'
poIMR0084	Fluorophore	TCC TGT GTC CTC CCC CAG TAG ATA
poIMR0085		CTT AAT GAG GGG CCG GTT GA
poIMR0086		GAC ATG GCC CCA CAA

Reaction A

Component	Final Concentration
ddH2O
Kapa 2G HS buffer	1.00
MgCl2	2.00
dNTPS-kapa	0.20
poIMR0084	0.50
poIMR0085	0.50
Glycerol	5.00
Kapa 2G HS taq polym	0.01
DNA

Cycling

Step	Temp °C	Time	Note
1	94.0	--
2	94.0	--
3	65.0	--	-0.5 C per cycle decrease
4	68.0	--
5		--	repeat steps 2-4 for 10 cycles
6	94.0	--
7	60.0	--
8	72.0	--
9		--	repeat steps 6-8 for 28 cycles
10	72.0	--
11	10.0	--	hold

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
006807	B6Ei.129S6(Cg)-Gata4^tm1.1Sho/EiJ
006808	D2.129S6(Cg)-Gata4^tm1.1Sho/EiJ
2 strains use this protocol