For in-depth product & services help, ask our
Technical Information Scientists
Stock No: 029100
Protocol 17920: Standard PCR Assay - Fxn<em#Lutzy>-KO
Version 1.0
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 = 297 bp
Heterozygote = 198 bp and 297 bp
Wild type = 198 bp
Ignore floxed band at ~250 bp for scoring purposes.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 26219 | GTA GAC GTG GGG ATC AGT CC | Common | A | |||
| 26220 | CGC TCT GCT TTT TGA TGT TC | Wild type Reverse | A | |||
| 26221 | ACT CAC ATC ATG GTG GAA AGC | Mutant Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 26219 | 0.50 uM |
| 26220 | 0.50 uM |
| 26221 | 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 |
|---|---|
| 028040 | B6(Cg)-Fxnem2.1Lutzy/J |
| 029100 | B6.Cg-Fxnem2.1Lutzy Tg(Ckmm-cre)5Khn/J |
| 029720 | B6.Cg-Fxnem2Lutzy Fxnem2.1Lutzy Tg(Ckmm-cre)5Khn/J |
| 030218 | B6.Cg-Pvalbtm1(cre)Arbr Fxnem2.1Lutzy/J |
| 029721 | B6.Cg-Pvalbtm1(cre)Arbr Fxnem2Lutzy Fxnem2.1Lutzy/J |
| 031007 | STOCK Fxnem2.1Lutzy Tg(FXN)Y47Pook/J |
| 030395 | STOCK Fxnem2.1Lutzy Tg(FXN)YG8Pook/800J |
| 030930 | STOCK Fxnem2.1Lutzy Tg(FXN)YG8Pook/J |
| 8 strains use this protocol | |