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Stock No: 029721
Protocol 26297: Standard PCR Assay - Fxn<tm1Mkn>alternate3
Version 1.2
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
Mutant = 226 bp
Heterozygote = 218 bp and 226 bp
Wild type = 218 bp
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 19125 | CTT CCC TCT ACC CTG CCT TC | Wild type Forward | A | |||
| 19126 | GGA GAA CAG TGG ACA CAG TAA CA | Common | A | |||
| oIMR8444 | GCC TGA AGA ACG AGA TCA GC | Mutant Forward | A | pPNT polyA |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 19125 | 0.50 uM |
| 19126 | 0.50 uM |
| oIMR8444 | 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 |
|---|---|
| 016842 | B6.129(Cg)-Fxntm1Mkn/J |
| 012329 | B6.Cg-Fxntm1.1Pand Fxntm1Mkn/J |
| 014162 | B6.Cg-Fxntm1Mkn Fxntm1Pand/J |
| 012910 | B6.Cg-Fxntm1Mkn Tg(FXN)YG22Pook/J |
| 012253 | B6.Cg-Fxntm1Mkn Tg(FXN)YG8Pook/J |
| 029721 | B6.Cg-Pvalbtm1(cre)Arbr Fxnem2Lutzy Fxnem2.1Lutzy/J |
| 008586 | B6.Cg-Tg(FXN*)1Sars Fxntm1Mkn/J |
| 031007 | STOCK Fxnem2.1Lutzy Tg(FXN)Y47Pook/J |
| 024097 | STOCK Fxntm1Mkn Tg(FXN)Y47Pook/J |
| 024113 | STOCK Fxntm1Mkn Tg(FXN)YG8Pook/2J |
| 010963 | STOCK Fxntm1Mkn Tg(FXN)YG22Pook/J |
| 008398 | STOCK Fxntm1Mkn Tg(FXN)YG8Pook/J |
| 030324 | STOCK Tg(FXN)YG8Pook/J |
| 018299 | STOCK Tg(FXN*)1Sars Fxntm1Mkn/J |
| 14 strains use this protocol | |