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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 = 148 bp
Heterozygote = 148 bp and 224 bp
Wild type = 224 bp
Gel on 3% due to nonspecific bands close to wt.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 24463 | AAT TTT TCC CTC CGC AGA CT | Common | A | |||
| 24464 | ACA GCT GCT CAG GGC TAT TG | Wild type Reverse | A | |||
| 24465 | AGG AGG TTG AGG TGA GGA TG | 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 |
| 24463 | 0.50 uM |
| 24464 | 0.50 uM |
| 24465 | 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 |
|---|---|
| 038105 | B6(Cg)-Apoetm1.1(APOE*4)Adiuj Isr(HSA17)1Mdk Appem2Aduci/AduciJ |
| 038104 | B6(Cg)-Apoetm1.1(APOE*4)Adiuj Isr(HSA17)1Mdk Apptm1.1Aduci/AduciJ |
| 034455 | B6(SJL)-Apoeem2(APOE*)Adiuj Appem1Adiuj/J |
| 039777 | B6(SJL)-Apoeem3(APOE*4)Msasn/J |
| 034415 | B6(SJL)-Apoetm1.1(APOE*4)Adiuj Appem1Adiuj/J |
| 034454 | B6(SJL)-Apoetm1.1(APOE*4)Adiuj Plcg2em3Adiuj Appem1Adiuj Trem2em1Adiuj/J |
| 028709 | B6(SJL)-Apoetm1.1(APOE*4)Adiuj Trem2em1Adiuj/J |
| 037778 | B6(SJL)-Apoetm1.1(APOE*4)Adiuj Isr(HSA17)2Mdk Appem1Adiuj/J |
| 027894 | B6(SJL)-Apoetm1.1(APOE*4)Adiuj/J |
| 031668 | B6(SJL)-Cr2tm1(CR2,CR1)How Apoetm1.1(APOE*4)Adiuj Trem2em1Adiuj/J |
| 031464 | B6(SJL)-Mthfrem1Adiuj/J |
| 034545 | B6(SJL)-Tardbpem9Lutzy Apoetm1.1(APOE*4)Adiuj Maptem2Lutzy/KmsoJ |
| 033868 | B6.Cg-Adamts4em1Adiuj Apoetm1.1(APOE*4)Adiuj Appem1Adiuj Trem2em1Adiuj/J |
| 039776 | B6.Cg-Apoeem1(APOE*)Msasn/J |
| 029018 | B6.Cg-Apoeem2(APOE*)Adiuj/J |
| 029017 | B6.Cg-Apoeem3(APOE*)Adiuj/J |
| 039778 | B6.Cg-Apoeem4(APOE*)Msasn/J |
| 030320 | B6.Cg-Apoetm1.1(APOE*4)Adiuj Abca7em1Adiuj Trem2em1Adiuj/J |
| 036243 | B6.Cg-Apoetm1.1(APOE*4)Adiuj Abca7em2Adiuj Appem1Adiuj Trem2em1Adiuj/J |
| 030283 | B6.Cg-Apoetm1.1(APOE*4)Adiuj Abca7em2Adiuj Trem2em1Adiuj/J |
| 033873 | B6.Cg-Apoetm1.1(APOE*4)Adiuj Appem1Adiuj Cd2apem1Adiuj Trem2em1Adiuj/J |
| 033869 | B6.Cg-Apoetm1.1(APOE*4)Adiuj Appem1Adiuj Trem2em1Adiuj Bin1em1Adiuj/J |
| 030670 | B6.Cg-Apoetm1.1(APOE*4)Adiuj Appem1Adiuj Trem2em1Adiuj/J |
| 031722 | B6.Cg-Apoetm1.1(APOE*4)Adiuj Appem2Adiuj Trem2em1Adiuj/J |
| 039775 | B6.Cg-Apoetm1.1(APOE*4)Adiuj Apptm1.1Dnli/MsasnJ |
| 030673 | B6.Cg-Apoetm1.1(APOE*4)Adiuj Ceacam1em1Adiuj Trem2em1Adiuj/J |
| 031944 | B6.Cg-Apoetm1.1(APOE*4)Adiuj Clasp2em1Adiuj Trem2em1Adiuj/J |
| 030304 | B6.Cg-Apoetm1.1(APOE*4)Adiuj Il1rapem1Adiuj Trem2em1Adiuj/J |
| 032777 | B6.Cg-Apoetm1.1(APOE*4)Adiuj Il1rapem2Adiuj Appem1Adiuj Trem2em1Adiuj/J |
| 033541 | B6.Cg-Apoetm1.1(APOE*4)Adiuj Il34em1Adiuj Appem1Adiuj Trem2em1Adiuj/J |
| 033770 | B6.Cg-Apoetm1.1(APOE*4)Adiuj Meox2tm1(cre)Sor Trem2em1Adiuj/J |
| 030674 | B6.Cg-Apoetm1.1(APOE*4)Adiuj Plcg2em3Adiuj Trem2em1Adiuj/J |
| 033913 | B6.Cg-Apoetm1.1(APOE*4)Adiuj Ptk2bem1Adiuj Appem1Adiuj Trem2em1Adiuj/J |
| 033867 | B6.Cg-Apoetm1.1(APOE*4)Adiuj Ptprbem1Adiuj Appem1Adiuj Trem2em1Adiuj/J |
| 032775 | B6.Cg-Apoetm1.1(APOE*4)Adiuj Scimpem1Adiuj Appem1Adiuj Trem2em1Adiuj/J |
| 031942 | B6.Cg-Apoetm1.1(APOE*4)Adiuj Snx1em1Adiuj Trem2em1Adiuj/J |
| 031940 | B6.Cg-Apoetm1.1(APOE*4)Adiuj Sorl1em1Adiuj Trem2em1Adiuj/J |
| 033875 | B6.Cg-Epha1em1Adiuj Apoetm1.1(APOE*4)Adiuj Appem1Adiuj Trem2em1Adiuj/J |
| 031938 | B6.Cg-Kif21bem1Adiuj Apoetm1.1(APOE*4)Adiuj Trem2em1Adiuj/J |
| 036242 | B6.Cg-Mthfrem1Adiuj Apoetm1.1(APOE*4)Adiuj Appem1Adiuj Trem2em1Adiuj/J |
| 030922 | B6.Cg-Mthfrem1Adiuj Apoetm1.1(APOE*4)Adiuj Trem2em1Adiuj/J |
| 034543 | B6.Cg-Tardbpem9Lutzy Apoetm1.1(APOE*4)Adiuj Appem5Msasn/KmsoJ |
| 034546 | B6.Cg-Tardbpem9Lutzy Apoetm1.1(APOE*4)Adiuj Maptem2Lutzy Psen1em1Msasn Appem5Msasn/KmsoJ |
| 034544 | B6.Cg-Tardbpem9Lutzy Apoetm1.1(APOE*4)Adiuj Psen1em1Msasn Appem5Msasn/KmsoJ |
| 034542 | B6.Cg-Tardbpem9Lutzy Apoetm1.1(APOE*4)Adiuj Psen1em1Msasn/KmsoJ |
| 039997 | B6.Cg-Tardbpem9Lutzy Sncaem2Lutzy Apoetm1.1(APOE*4)Adiuj Maptem2Lutzy Psen1em1Msasn Appem5Msasn/KmsoJ |
| 031946 | B6;SJL-Apoetm1.1(APOE*4)Adiuj Erc2em1Adiuj Trem2em1Adiuj/J |
| 031950 | B6;SJL-Apoetm1.1(APOE*4)Adiuj Mtmr4em1Adiuj Trem2em1Adiuj/J |
| 030771 | B6;SJL-Apoetm1.1(APOE*4)Adiuj Plcg2em1Msasn Trem2em1Adiuj/J |
| 031952 | B6;SJL-Apoetm1.1(APOE*4)Adiuj Shc2em1Adiuj Trem2em1Adiuj/J |
| 031948 | B6;SJL-Slc6a17em1Adiuj Apoetm1.1(APOE*4)Adiuj Trem2em1Adiuj/J |
| 51 strains use this protocol | |