Protocol 24598: Separated MCA Assay - Gt(ROSA)26Sor<tm1sor> STD
Version 5.2

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

Melt Peaks
Wt = 88°C +/- 1
Mut =
88°C +/- 1

Mutant = 340 bp
Heterozygote = 340 bp and 650 bp
Wild type = 650 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR8052 GCG AAG AGT TTG TCC TCA ACC Mutant A SA site
oIMR8545 AAA GTC GCT CTG AGT TGT TAT Common A, B ROSA26
oIMR8546 GGA GCG GGA GAA ATG GAT ATG Wild type B ROSA26

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTPS-kapa 0.26 mM
oIMR8052 0.50 uM
oIMR8545 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.

Reaction B

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTPS-kapa 0.26 mM
oIMR8545 0.50 uM
oIMR8546 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
009582 B6(129S4)-Et(cre/ERT2)1645Rdav/J
009585 B6(129S4)-Et(cre/ERT2)2047Rdav/J
012687 B6(129S4)-Tg(SYN1-icre/mRFP1)9934Rdav/J
012580 B6.129P2(Cg)-Hprttm59(Ple123-lacZ)Ems/Mmjax
012581 B6.129P2(Cg)-Hprttm62(Ple153-lacZ)Ems/Mmjax
012583 B6.129P2(Cg)-Hprttm68(Ple127-lacZ)Ems/Mmjax
013586 B6.129P2-Gt(ROSA)26Sortm1Nik/J
003474 B6.129S4-Gt(ROSA)26Sortm1Sor/J
007743 B6.129S4-Gt(ROSA)26Sortm3(phiC31*)Sor/J
014602 B6.Cg-Gt(ROSA)26Sortm1(rtTA*M2)Jae Col1a1tm1(tetO-mCherry)Eggn/J
023749 B6.Cg-Gt(ROSA)26Sortm1(rtTA*M2)Jae Tg(tetO-Pou5f1,-Sox2,-Klf4,-Myc)1Srn/J
010527 B6;129-Gt(ROSA)26Sortm1(DTA)Mrc/J
008883 B6;129-Gt(ROSA)26Sortm1(SNCA*A53T)Djmo/TmdJ
008889 B6;129-Gt(ROSA)26Sortm2(SNCA*119)Djmo/TmdJ
022516 B6;129S-Gt(ROSA)26Sortm1(Cdkn1c)Jfpa/J
003309 B6;129S4-Gt(ROSA)26Sortm1Sor/J
019101 B6N.129S4(B6)-Gt(ROSA)26Sortm1Sor/CjDswJ
019013 B6N.129S6(Cg)-Gt(ROSA)26Sortm2(EGFP/cre)Alj/J
007900 C57BL/6-Gt(ROSA)26Sortm1(HBEGF)Awai/J
011011 STOCK Gt(ROSA)26Sortm1(rtTA*M2)Jae Col1a1tm4(tetO-Pou5f1,-Sox2,-Klf4,-Myc)Jae/J
011013 STOCK Gt(ROSA)26Sortm1(rtTA*M2)Jae Col1a1tm5(tetO-Pou5f1,-Klf4,-Myc)Jae/J
018903 STOCK Gt(ROSA)26Sortm2(EGFP/cre)Alj/J
012584 STOCK Hprttm69(Ple134-lacZ)Ems/Mmjax
23 strains use this protocol