Stock No: 027993
Protocol 18045: Sanger sequencing Assay - Comt<tm#(COMT)Xzho>-SEQ-Alternate 1
Version 1.0

Notes

Mut = A

WT= G

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

Sequence

CGAGATCAACCCCGACTGTGCCGCCATCACCCAGCGGATGGTGGATTTCGCTGGC(g/a)TGAAGGACAAG

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
26417 CGT GAT TCA GGA GCA CCA Forward A human COMT
26418 TCC AAG AGA AGC GTG TCC Reverse A human COMT

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTPS-kapa 0.26 mM
26417 0.50 uM
26418 0.50 uM
Glycerol 6.50 %
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
027990 129-Comttm1(COMT)Xzho/J
027993 129-Comttm2(COMT)Xzho/J
032382 B6.129-Comttm1(COMT)Xzho/J
032383 B6.129-Comttm2(COMT)Xzho/J
4 strains use this protocol