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Stock No: 005491
Protocol 37868: Probe Assay - Mapt<tm1(EGFP)Klt>-Probe
Version 1.0
Notes
Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
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
>chr11:104282410+104282510 101bp AATGGAAGACCATGCTGGAG TGGCCAGTTGTGTATGTCCA
Mut= 93 bp
Wt= 101 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence:
TCGCCAGGAGTTTGACACAATGGAAGACCATGCTGGAGATTACACTCTGCTCCAAGACCAAGAAGGAGACATGGACCATGGCTTAAAAGgtcagtggggtggacatacacaactggccagtcacagtg
Mutant Sequence:
ACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAAC
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 12390 | GTC TTG TAG TTG CCG TCG TC | Mutant Reverse | A | |||
| 39873 | TGG CCA GTT GTG TAT GTC CA | Wild type Reverse | A | |||
| 52360 | CAT GAA GCA GCA CGA CTT CT | Mutant Forward | A | |||
| 52361 | Fluorophore-1 | AAG GAG ACA TGG ACC ATG GCT | Quencher-1 | WT Probe | ||
| 52362 | Fluorophore-2 | CGA AGG CTA CGT CCA GGA GC | Quencher-2 | MUT Probe | ||
| oIMR7824 | AAT GGA AGA CCA TGC TGG AG | Wild type Forward | A |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 12390 | 0.40 uM |
| 39873 | 0.40 uM |
| 52360 | 0.40 uM |
| oIMR7824 | 0.40 uM |
| Wt Probe | 0.15 uM |
| Mutant Probe | 0.15 uM |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 95.0 | -- | |
| 2 | 95.0 | -- | |
| 3 | 60.0 | -- | |
| 4 | -- | repeat steps 2-3 for 40 cycles | |
| 5 | 40.0 | -- | Forever |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.