Protocol 27253: Sanger sequencing Assay - Tg(Rho*)Rieke SEQ
Version 1.2

Notes

27645 GACGACGCCTCTGC(CA/GG)CCG(C/T)TTCCAAG(A/G)C(G/C)GAG(A/G)CC(AG/GC)
27646 GACGACGCCTCTGCCAC(c/g)g(ct/tc)tccaagacggagacc(ag/gc)
27644 GACGACGCC(T/G)C(T/G)GCC(a/g)ccg(c/t)ttccaag(a/g)cggag(a/g)cc
27649 and 27648 GACGACGCCTC(t/g)gcc(a/g)ccg(c/t)ttccaag(a/g)cgga(a/g)
27647  GACGACGC(ct/gg)c(t/c)gccaccg(c/t)t(t/g)ccaagacggagacc(ag/gc)

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

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
24408 GCT TAG TGA GGG GAC ATG CT Forward A
24412 ACT GAG GGA GCC TGC ATG A Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTPS-kapa 0.26 mM
24408 0.50 uM
24412 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
027644 STOCK Rhotm1Jlem Tg(Rho*)1PRieke/J
027645 STOCK Rhotm1Jlem Tg(Rho*)2PRieke/J
027646 STOCK Rhotm1Jlem Tg(Rho*)5PRieke/J
027647 STOCK Rhotm1Jlem Tg(Rho*)STM2Rieke/J
027648 STOCK Rhotm1Jlem Tg(Rho*)TTM1Rieke/J
027649 STOCK Rhotm1Jlem Tg(Rho*)TTM4Rieke/J
6 strains use this protocol