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Notes
validation/LC Conversion data attached to rec 169358
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
Wt=410 bp
Mut=110 bp
Two public protocols are supplied- different band sizes.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| oIMR3439 | TTT TCT CCC TCT TCA GAG TGA T | Common | A | |||
| oIMR3440 | CTG TTT CAA GGG AGT TGT GGC | Wild type Reverse | A | |||
| oIMR7210 | GGT AAC GCC AGG GTT TTC C | Mutant Reverse | A | LacZ |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| oIMR3439 | 0.50 uM |
| oIMR3440 | 0.50 uM |
| oIMR7210 | 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 |
|---|---|
| 008629 | B6.Cg-Tg(SMN2)11Tro Smn1tm1Msd/J |
| 008631 | B6.Cg-Tg(SMN2)11Tro Tg(SMN2)46Tro Smn1tm1Msd/J |
| 008630 | B6.Cg-Tg(SMN2)46Tro Smn1tm1Msd/J |
| 006385 | B6;129P2-Syt1tm3Sud/J |
| 006386 | B6;129P2-Syt1tm5Sud/J |
| 006214 | FVB.129P2-Smn1tm1Msd/J |
| 008782 | FVB.Cg-Grm7Tg(SMN2)89Ahmb Tg(SMN2*A111G)588Ahmb Smn1tm1Msd/J |
| 008209 | FVB.Cg-Grm7Tg(SMN2)89Ahmb Smn1tm1Msd Tg(ACTA1-SMN)69Ahmb/J |
| 005026 | FVB.Cg-Grm7Tg(SMN2)89Ahmb Smn1tm1Msd Tg(SMN1*A2G)2023Ahmb/J |
| 009134 | FVB.Cg-Grm7Tg(SMN2)89Ahmb Smn1tm1Msd Tg(SMN2*A111G)591Ahmb/J |
| 005025 | FVB.Cg-Grm7Tg(SMN2)89Ahmb Smn1tm1Msd Tg(SMN2*delta7)4299Ahmb/J |
| 031906 | FVB.Cg-Grm7Tg(SMN2)89Ahmb Smn1tm1Msd Tg(SMN2-SMN1*T274I)5Ahmb/J |
| 005024 | FVB.Cg-Grm7Tg(SMN2)89Ahmb Smn1tm1Msd/J |
| 030214 | FVB.Cg-Grm7Tg(SMN2)89Ahmb Smn1tm1Msd Tg(Prnp-PLS3)14Ahmb Tg(SMN2*delta7)4299Ahmb/J |
| 030970 | FVB.Cg-Mapk10tm1Flv Grm7Tg(SMN2)89Ahmb Smn1tm1Msd Tg(SMN2*delta7)4299Ahmb/LganJ |
| 034287 | FVB.Cg-Smn1tm1Msd Tg(SMN2)1Cdid/J |
| 008206 | FVB.Cg-Smn1tm1Msd Tg(SMN2)566Ahmb/J |
| 031909 | FVB.Cg-Tg(SMN2*A111G)588Ahmb Tg(SMN2-SMN1*Q282A)2Ahmb Smn1tm1Msd/J |
| 031907 | STOCK Grm7Tg(SMN2)89Ahmb Tg(SMN2-SMN1*Q282A)2Ahmb Smn1tm1Msd/J |
| 008203 | STOCK Grm7Tg(SMN2)89Ahmb Smn1tm1Msd Tg(ACTA1-SMN)63Ahmb/J |
| 032003 | STOCK Grm7Tg(SMN2)89Ahmb Smn1tm1Msd Tg(Hlxb9-GFP)1Tmj Tg(SMN2*delta7)4299Ahmb/J |
| 006570 | STOCK Grm7Tg(SMN2)89Ahmb Smn1tm1Msd Tg(Hlxb9-GFP)1Tmj/J |
| 008212 | STOCK Grm7Tg(SMN2)89Ahmb Smn1tm1Msd Tg(Prnp-SMN)92Ahmb/J |
| 025102 | STOCK Grm7Tg(SMN2)89Ahmb Smn1tm1Msd Tg(Rnu7-SMN2*,PGK1-EGFP)4Dasch/DaschMmjax |
| 018916 | STOCK Grm7Tg(SMN2)89Ahmb Smn1tm1Msd Tg(SMN1-SMN2*)16Cll/CllJ |
| 031908 | STOCK Grm7Tg(SMN2)89Ahmb Smn1tm1Msd Tg(SMN2-SMN1*D44V)10Ahmb/J |
| 017596 | STOCK Gt(ROSA)26Sortm1.1(rtTA,EGFP)Nagy Grm7Tg(SMN2)89Ahmb Smn1tm1Msd Tg(SMN2*delta7)4299Ahmb Tg(tetO-SMN2,-luc)#aAhmb/J |
| 017597 | STOCK Gt(ROSA)26Sortm1.1(rtTA,EGFP)Nagy Grm7Tg(SMN2)89Ahmb Smn1tm1Msd Tg(SMN2*delta7)4299Ahmb Tg(tetO-SMN2,-luc)#bAhmb/J |
| 28 strains use this protocol | |