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Protocol 24258: Standard PCR Assay - Smn1<tm3(SMN2/Smn1)Mrph>
Version 5.2
Notes
Melting curve analysis is done using a Roche Light Cycler 480.
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
Evagreen
Multiplex-HT-TD 65-60 RS Macro TmCalling analysis
WT@88
Mut@81.5
Mutant = 103bp
Heterozygote = 103bp and 143bp
Wild type = 143bp
Some strains are Hom Lethal
JR 14562- Smn1 tm3 will not see hets in stains that include the Smn1 tm5 allele. Results for the tm3 allele will be wild or hom.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| oIMR7033 | TGG GAG TCC ATC CAT CCT AAG TC | Wild type Forward | A | |||
| oIMR7034 | GCT AAG AAA ATG ACA ATT GCA CAT TTG | Wild type Reverse | A | |||
| oIMR7035 | GGC AGT TTT AGA CTC ATC ATG TAT CTG | Mutant Forward | A | |||
| oIMR7036 | ACT TAT GGA GAT CCC TCG AGA TAA C | 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 |
| oIMR7033 | 0.50 uM |
| oIMR7034 | 0.50 uM |
| oIMR7035 | 0.50 uM |
| oIMR7036 | 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 |
|---|---|
| 007966 | B6.Cg-Smn1tm3(SMN2/Smn1)Mrph/J |
| 007249 | B6;129-Smn1tm3(SMN2/Smn1)Mrph/J |
| 008704 | B6;129-Smn1tm6(SMN2)Mrph/J |
| 007964 | FVB.Cg-Smn1tm3(SMN2/Smn1)Mrph/J |
| 008783 | STOCK Grm7Tg(SMN2)89Ahmb Smn1tm3(SMN2/Smn1)Mrph Tg(CAG-cre/Esr1*)5Amc Tg(SMN2*delta7)4299Ahmb/J |
| 007951 | STOCK Grm7Tg(SMN2)89Ahmb Smn1tm3(SMN2/Smn1)Mrph Tg(SMN2*delta7)4299Ahmb/J |
| 6 strains use this protocol | |