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Notes
This assay will NOT distinguish hemizygous from homozygous transgenic animals.
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
Transgene = 187 bp
Internal positive control = 415 bp
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 21238 | CTG TCC CTG TAT GCC TCT GG | Internal Positive Control Forward | A | |||
| 21239 | AGA TGG AGA AAG GAC TAG GCT ACA | Internal Positive Control Reverse | A | |||
| 24712 | CTT GGA ATA AGG CCG GTG T | Transgene Forward | A | |||
| 24713 | AGC TTC CAG AGG AAC TGC TTC | Transgene Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 21238 | 0.50 uM |
| 21239 | 0.50 uM |
| 24712 | 0.50 uM |
| 24713 | 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 |
|---|---|
| 039195 | B6(Cg)-Il17atm1.1(EYFP/icre)Ehs/J |
| 031824 | B6(FVB)-Tlr4tm1.1Gbrt/J |
| 033394 | B6.129-Ntsr2tm1.1(cre)Giml/J |
| 031559 | B6.Cg-Crhtm1.1(flpo)Bsab/J |
| 030211 | B6.Cg-Npytm1.1(flpo)Hze/J |
| 017580 | C.129S4(B6)-Ifngtm3.1Lky/J |
| 032131 | C57BL/6-Tg(Scx-GFP/cre)1Stzr/J |
| 012899 | STOCK Agrptm1(cre)Lowl/J |
| 031289 | STOCK Lmx1btm2(cre/ERT2)Rjo/J |
| 029803 | STOCK Mki67tm2.1(cre/ERT2)Cle/J |
| 029429 | STOCK Or8a1tm26Mom Tg(Or8a1,-MAPT/mCherry)5MGaFeins/J |
| 006677 | STOCK Or8a1tm28(cre)Mom/MomJ |
| 036906 | STOCK Tmem178btm1c(KOMP)Wtsi Tmem178tm1.1Sud/J |
| 026703 | STOCK Vsx1tm2Mci/ChowJ |
| 026707 | STOCK Tg(Grm6-lacZ)1Chow/J |
| 018281 | STOCK Tg(Wnt7a-EGFP/cre)#Bhr/Mmjax |
| 16 strains use this protocol | |