Protocol 3212: Restriction Enzyme Digest Assay - Tg(YACW408A5)1952Ricc
Version 2.0

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

Transgene = 400 bp
Internal positive control = 449 bp
Separate digested products on a 3.0% agarose gel.**

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR5265 TTC AGA TCT GAC CTC AGA CCC
oIMR5266 AGC TTC TTG ATC GCA CCG TTC

Reaction A

Component Final Concentration
ddH2O
AB PCR BufferII 1.00 X
MgCl2 2.00 mM
dNTP 0.20 mM
oIMR5265 1.00 uM
oIMR5266 1.00 uM
DNA Loading Dye 0.69 mM
Taq DNA Polymerase 0.03 U/ul
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 58.0 --
4 72.0 -- repeat steps 2-4 for 35 cycles
5 72.0 --
6 10.0 -- hold**
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Reaction B

Component Final Concentration
ddH2O
AB PCR BufferII 5.45 X
BsoB1

Cycling

Step Temp °C Time Note
1 37.0 -- Add 2.2 ul of buff/Enzyme to entire PCR product
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.