Genotyping Protocols Database
Stock No: 026195
Protocol 26831: Sanger sequencing Assay - Prkra<lear-3J>-SEQ
Version 1.2

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

Mut = A/A

WT= G/G

Sequence

 

ttcagtagcgagccagcacacgtaaagtgacattgttttgcattatggccgcggatatgaaa
cacatacttgttcacagagactaagaacttcttcctcccgTCTTTAAtgcagGTGAAGGTAC
AAGTAAGAAGCTGGCGAAGCACAGAGCTGCGGAGGCCGCCATAAACATTTTGAAAGCCAATG
CAAGTATTTGgtGa(g/a)cTaaatttctttgtattctgcagctcaaaattttcatggctgaggtaa
gatgaaaatttttcatatgCTCTCAAaagcgacagagcggacagttgt

 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
21232 TTC AGT AGC GAG CCA GCA C Forward A
21233 ACA ACT GTC CGC TCT GTC G Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTPS-kapa 0.26 mM
21232 0.50 uM
21233 0.50 uM
Glycerol 6.50 %
Dye 1.00 X
Kapa 2G HS taq polym 0.03 U/ul
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 (Touchdown)
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.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.

Strains Using This Protocol

This is the only strain that uses this protocol.