Stock No: 013110
Protocol 5885: Pyrosequencing Assay - Uchl1<gad-2J>
Version 1.0

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

Sequence

GGTACCATCGGGTTGATCCACG(c/a)AGTGGCCAACAACCAAGACAAGCTGG

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
11485 GCA GAC CAT CGG AAA CTC CT Forward
11486 Fluorophore CGT GGC ATG TAC CCA CAA TAA TA Reverse
11487 CAT CGG GTT GAT CCA SEQ

Reaction A

Component Final Concentration
ddH2O
KAPA LR HotStart Buf Buffer does not contain MgCl2. 0.50
MgCl2 2.00
dNTP 0.13
KAPA LR HotStart Taq Initial Denature 3 Min This is a two enzyme system. One with proofreeading the other without. HotStart is provided by antibody. 0.01
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 60.0 --
4 65.0 -- Go to step 2-4, 50 times
6 65.0 --
7 10.0 -- hold
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

This is the only strain that uses this protocol.