Protocol 13397: Pyrosequencing Assay - Ins2<Akita>

C57BL/6-Ins2^Akita/J

Stock No: 003548

Version 4.0

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 = T/T
Heterozygote = C/T
Wild type = C/C

Sequence

GCTCTTCTCTATCCAGCTAGAAGCATCAGGGGTGGGGGTCATGATCCTCCCGGACAACT
CAGGGTGGGGGTTATGGGCTACCTGACGGTATTGGTTATCTTGGGGAAAGGAACTGTCT
ATGTTCCAGGTTTGGTCTAACAGGTACCTGTGGGAGGTGCCCAGGGAGCCAGGAGAAA
ACTGGGGATGCAGAGGGAACCAAAGAAGTCTTGGGTAGATCTTTTAGGGGCTATTGTGG
CTAGGCAGATAGATAGATTAAGCTGTGGCTACCCTACCAGCCCCACTCATGTCTCCTGGT
CCCACATATGCACATGCACATACACACACAACTTGTGCTCATTCAAAGGTTTTATTCATT
GCAGAGGGGTAGGCTGGGTAGTGGTGGGTCTAGTTGCAGTAGTTCTCCAGCTGGTAGA
GGGAGCAGATGCTGGTG(C/T)AGCACTGATCTACAATGCCACGCTTCTGCTGGGCCACC
TCCAGTGCCAAGGTCTGAAGGTCACCTGCTCCCGGGCCTCCACCCAGCTCCAGTTGTG
CCACTGCCAGGGAGGTTGTGTCAGAGCAGGCCAGGGCATCAGCAGCACAGAAGCAAA
GACACATAGGAAAATCCAGTCAGGAGTAGCTGAGCAACCTCCTACTACCCCAGGTTTTC
TCGCCCCTTAACCCCTGCAGGTGGCAGGGGTTGCATGCCTGTGGGACAAGTCACTGGTC
ATGGATGGACACATGGGCCTCTAAAGCCTACTCATCTTCAGTCAGTGCAGTGCTCTATGA
GGGCCCTAAATAGGTACCTATCCTGGGATCTAGTCACATGTCCCAAGTTACTACCACCAA
GGACCCCTTAAGGCAGGATTCTTACTCATTCCTGGCAGCTGCCATGTTTACATATGTCTAG
CTTCCCCCAGAAACGTGTCCCCACTCCCTGGTTATCCCGAGACCCTCCACACCTAGGACA
CAATTTACAATGTCCCAAGTGTGAAGAAAACCAGGGTAGTTAGCACTGGGGACAG

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction	Note
poIMR0041		TGG GTC TAG TTG CAG TAG TTC TCC
poIMR0042	Fluorophore	GAA GCG TGG CAT TGT AGA TCA GT
poIMR0043		GAG CAG ATG CTG GTG

Reaction A

Component	Final Concentration
ddH2O
Kapa 2G HS buffer	1.00
MgCl2	2.00
dNTPS-kapa	0.20
poIMR0041	0.50
poIMR0042	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
012307	B6.129-Apoe^tm3(APOE*4)Mae Ldlr^tm1(LDLR)Mae/J
006580	B6.Cg-Ins2^Akita Ldlr^tm1Her/J
003548	C57BL/6-Ins2^Akita/J
008659	NOD.Cg-Rag1^tm1Mom Ins2^Akita Prf1^tm1Sdz/SzJ
014568	NOD.Cg-Rag1^tm1Mom Ins2^Akita Il2rg^tm1Wjl/SzJ
5 strains use this protocol