Stock No: 017515
Protocol 24057: Standard PCR Assay - Pdcd1lg2<tm1Dmp>
Version 1.2

Notes

This assay does not work well without the use of a Hotstart Taq polymerase.

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 =  165 bp
Heterozygote = 165 bp and 296 bp
Wild type = 296 bp

 

<p><span style="color: #ff0000">Use 3% gel</span></p>

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
13367 TCC TGT AGC AGG TAG GAA CGG AAG Wild type Forward A
13368 TGC CTC AGT CAC TGT CCC TGA TAG Wild type Reverse A
13369 TCT ATA TCA TGG CCG ACA AGC AGA Mutant Forward A
13370 ACT GGG TGC TCA GGT AGT GGT TGT Mutant Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
13367 0.50 uM
13368 0.50 uM
13369 0.50 uM
13370 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.