Stock No: 002207
Protocol 27075: Standard PCR Assay - Ldlr<tm1Her>-alternate 1
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

Mutant = ~179 bp
Heterozygote = ~179 bp and 351 bp
Wild type = 351 bp

Mut is GC rich

 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
19799 TAT GCA TCC CCA GTC TTT GG Common A
19800 CTA CCC AAC CAG CCC CTT AC Wild type Reverse A
oIMR7770 ATA GAT TCG CCC TTG TGT CC 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
19799 0.50 uM
19800 0.50 uM
oIMR7770 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

Stock Number Strain Name
007070 AK.129S7(B6)-Ldlrtm1Her/J
006952 B6.129-Akt2tm1.1Mbb Ldlrtm1Her/J
002246 B6.129-Apoetm1Unc Ldlrtm1Her/J
006883 B6.129S7-Ldlrtm1Her Sod2tm1Leb/J
002207 B6.129S7-Ldlrtm1Her/J
006580 B6.Cg-Ins2Akita Ldlrtm1Her/J
022488 B6.Cg-Ldlrtm1Her Tg(Tagln-Pth1r*)2Towl/J
006877 B6.Cg-Ldlrtm1Her Tg(H2-K-AKR1B1)1Tj/J
006906 B6.Cg-Lepob Ldlrtm1Her/J
002245 B6;129-Apoetm1Unc Ldlrtm1Her/J
003000 B6;129S-Ldlrtm1Her Apobtm2Sgy/J
002465 B6;129S-Lrpap1tm1Her Ldlrtm1Her/J
002077 B6;129S7-Ldlrtm1Her/J
012845 CByJ.129S7(B6)-Ldlrtm1Her/J
007068 D2.129S7(B6)-Ldlrtm1Her/J
004192 STOCK Mttptm2Sgy Ldlrtm1Her Apobtm2Sgy Tg(Mx1-cre)1Cgn/J
16 strains use this protocol