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This assay will NOT distinguish heterozygous from wildtype animals.
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 5 µl on the gel. PCR products are separated on 3.5 % MetaPhor agarose gel with 0.5 x SYBR Green I Nucleic Acid Stain.
From DI: For JR12866 use C57BL/6J for wild type control = 124 bp, plt/plt = no band (or very faint band)
From DI: For JR12873 use BALB/cJ for wild type control = 116 bp, plt/plt = no band (or very faint band)
| Products in B6 background | Products in BALB background | |||
| plt/plt | none | plt/plt | none | |
| wt/plt | 124bp | wt/plt | 116bp | |
| wt/wt | 124bp | wt/wt | 116bp | |
This assay will NOT distinguish heterozygous from wildtype animals.
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 10892 | TTC AAA CTC ATG AGT CTA TGG GG | A | ||||
| 10893 | ATA TAC ACG TAG ACT CGC ACG C | A |
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTPS-kapa | 0.26 mM |
| 10892 | 0.50 uM |
| 10893 | 0.50 uM |
| Glycerol | 6.50 % |
| Dye | 1.00 X |
| Kapa 2G HS taq polym | 0.03 U/ul |
| DNA |
| 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 |