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Stock No: 008060
Protocol 23559: Standard PCR Assay - 008060 MIT markers 2 of 2 protocols
Version 2.2
Notes
THIS STRAIN NEEDS TO BE TYPED FOR FOUR MIT MARKERS.
FML Buffer: 500 mM KCl, 100 mM Tris HCl pH 8.3, 15 mM MgCl2, 0.01% Gelatin
Add 42 µl of loading buffer (12% ficoll 400, 0.2% bromophenol blue, 0.04 M EDTA) diluted 1:4 with TEN (10 mM Tris pH 8.0, 1 mM EDTA pH 8.0, 10 mM NaCl) to PCR reaction. Load 2 µl of D11Mit132, D11Mit314, D11Mit42 and 5 µl of D11Mit339 on the gel. PCR products are separated on 3.5 % MetaPhor agarose gel with 0.5 x SYBR Green I Nucleic Acid Stain.
FML Buffer: 500 mM KCl, 100 mM Tris HCl pH 8.3, 15 mM MgCl2, 0.01% Gelatin
Add 42 µl of loading buffer (12% ficoll 400, 0.2% bromophenol blue, 0.04 M EDTA) diluted 1:4 with TEN (10 mM Tris pH 8.0, 1 mM EDTA pH 8.0, 10 mM NaCl) to PCR reaction. Load 2 µl of D11Mit132, D11Mit314, D11Mit42 and 5 µl of D11Mit339 on the gel. PCR products are separated on 3.5 % MetaPhor agarose gel with 0.5 x SYBR Green I Nucleic Acid Stain.
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
D11Mit339: C57L/J ≈ 127 bp, NOD/ShiLtJ ≈ 145 bp
D11Mit42: C57L/J ≈ 118 bp, NOD/ShiLtJ ≈ 106 bp
D11Mit42: C57L/J ≈ 118 bp, NOD/ShiLtJ ≈ 106 bp
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| D11Mit339-L | TGG AAG ATC CCA GAA AGT AAG TG | A | ||||
| D11Mit339-R | CAT TAA AGC AAA CAC TCT GTC TCT G | A | ||||
| D11Mit42-L | ACT AGC CAT ATG GTT TCT GAT GG | B | ||||
| D11Mit42-R | GTA GCA GGG CTG TGA GCT TT | B |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTPS-kapa | 0.26 mM |
| D11Mit339-L | 0.50 uM |
| D11Mit339-R | 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.
Reaction B
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTPS-kapa | 0.26 mM |
| D11Mit42-L | 0.50 uM |
| D11Mit42-R | 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
This is the only strain that uses this protocol.