Stock No: 011054
Protocol 29728: Standard PCR Assay - Ctbp1<tm1Sor>
Version 4.2

Notes

This assay does not work well without use of Hotstart Taq Polymerase.

The primers are not very stable. New dilutions must be made frequently in order to keep assay working.
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

Mut=~500bp

Heterozygote-500bp and 583bp

WT=583bp

Run multiplexed assay on a 3% gel and let run out for a long time.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
9743 GAA GTA CCA GTA CAG GGG ACG A
9744 CCC CAG CTG ACT TGA TGT CG A
9745 GTT ATC GCC GCT CCC GAT TCG A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
9743 0.50 uM
9744 0.50 uM
9745 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.