Genotyping Protocols Database
Protocol 10364: Standard PCR Assay - Tg(Pnkd,-DsRed)445Ljp
Version 1.0

Notes

This assay will NOT distinguish hemizygous from homozygous transgenic animals.

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 = 324 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
16904 ACA TAC ATG CAG GAA AAC ATC C Transgene Reverse
16905 AGT GCA GTC GTA AAA GTC AGA AC Transgene Forward
oIMR7338 CTA GGC CAC AGA ATT GAA AGA TCT Internal Positive Control Forward
oIMR7339 GTA GGT GGA AAT TCT AGC ATC ATC C Internal Positive Control Reverse

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.00
MgCl2 2.00
dNTPS-kapa 0.20
Primer 1 0.50
Primer 2 0.50
10x Loading Dye 0.50
Kapa 2G HS taq polym 0.01
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 65.0 -- -1.5 C per cycle decrease
4 68.0 --
5 -- Go to step 2-4, 10 times
6 94.0 --
7 50.0 --
8 72.0 --
9 -- Go to step 6-8, 28 times
10 72.0 --
11 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
Kapa 2G HS buffer 1.00
MgCl2 2.00
dNTPS-kapa 0.20
Primer 3 0.50
Primer 4 0.50
10x Loading Dye 0.50
Kapa 2G HS taq polym 0.01
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 65.0 -- -1.5 C per cycle decrease
4 68.0 --
5 -- Go to Step 2-4, 10 times
6 94.0 --
7 50.0 --
8 72.0 --
9 -- Go to Step 6-8, 28 times
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
022146 B6.Cg-Tg(Pnkd*A7V*A9V,-DsRed)704Ljp/J
022147 B6.Cg-Tg(Pnkd,-DsRed)445Ljp/J
2 strains use this protocol