Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 118 bp
Wild Type = 154 bp
chr4:141193328+141193481 154bp GCCTACCAAAGATCACATTTCC CAACACAGAGAAAGGTACTCAGC
Wt Sequence (deletions in lower case): TCATGGAGCCCATGCTGCTCTCAGACTCTGTGTGTAGTTGAGGCTGCCCTTGGACTAAGTTATGACAGGGTTATACCCCACGGGACCTGTCACTTAATACACCTCCCGGAGACCACAGCACAGCGACACAGAATGGCTTGTAGTGTCCACCGTTCTTTCTGTGACCGTGAGACGTACTTGAGGTGATAGTCCTCTGTTACCTGACATTCTCACTAGCCTACCAAAGATCACATTTCCTAGGAGAATCCCACTGCCTGTGGGCCATTGTGGCACTGTtgtaaagttaagccaaggcaacttgggaccttttgaattgggtgcccattccactccactttcaaagaaagctgagtacctttctctgtgttgggtatgaagatgtataaatcaataatgggtctcttagacctgaaaacccagctccccgccccccccagcccccttacctttctgctgtgtgacagacattgcctctcttcttgtccttcccaggaaaggtgacctgtgggtgagctcccaaatcctaatcacggtgctgttgccttttcctagggtcctatccttcccccaaagctggggccactctggtcgtgtacaaggacttgctagtgctatttggtggctggacaaggccaagtccctatcccttacaccagccagagagattctttgatgaaattcacacgtactcgccgtctaaaaactggtaagccccaaagagaacccagTCTGTGTCTGGAGGAAATGGGTTTCATTGTGTTATGCTTTAGCTCTTTTGGGTGTGGTGGGCAGAGGATGGACGTCCGGAGCCTTCTCTCTTCTCGCCATGAGAATGCTAGAGATCAGTTCACTTTGTCAGGCTCCGTGGCAAGTGCCTTAGCCCACCAAGCCAGCTCGCTTCCCCCAAAGTGGGCTTTAAATCGATGGATTCTTTATTTCTGTGTATCAAGAAGCTCGCGG
This is a 452 bp deletion beginning at Chromosome 4 position 140,920,700 bp and ending after 140,921,151 bp (GRCm39/mm39).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
59539 | GCC TAC CAA AGA TCA CAT TTC C | Common | A | |||
59540 | CAA CAC AGA GAA AGG TAC TCA GC | Wild type Reverse | A | |||
59541 | Fluorophore-1 | AGC CAA GGC AAC TTG GGA CC | Quencher-1 | WT Probe | ||
59542 | CCA CAC CCA AAA GAG CTA AAG | Mutant Reverse | A | |||
59543 | Fluorophore-2 | ATT GTG GCA CTG TTC TGT GTC TGG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
59539 | 0.40 uM |
59540 | 0.40 uM |
59542 | 0.40 uM |
Wt Probe | 0.15 uM |
Mutant Probe | 0.15 uM |
DNA |
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 |