Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:134861364+134861460 97bp CTTTGTTTGACAGGACTTTTCC ACTGTTCTGCAGTCAGACATTCG
Mut= 97 bp
Wt= 97 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TTGACACATGACTCTAGGTTACAATGTTTACAAGAGATTTGATAGGTGTGTGAAATGAAGACCTAGACATAAGGTGAAAATAAGGGCTGGTATTTGTATTGTGATTGAAAGTTCAACAAATTCCAAGTCCTGTATATTTCATCCTATAagattctttagcgtgtgtgtgtatttctctgcaggtaccacatcattgttatcatcctggtgtactgtttcccattgctcatcatgggtgtcacctacaccatcgttggaattactctctggggaggagagatcccaggagacacctgtgacaagtaccatgagcagcttaaggctaaacgaaaggtatggtcccatttaactgaccagtataactgtcccaaataactgacccctataactcacaagttggtgcagactgggccacttccaaggaggaaggcactgactgatgttagaaatatgttctcatatctcataatctgaatcctcctttgtttgacaggacttttcccttgcataacctctttgttctccctggcaaacctcagcccttggAAGGTCTGCGAATGTCTGACTGCAGAACAGTTACATCCTTAAGGATTAGCTGTTTTGTCAGTCCCTGTTGTGGCCCATTCTCAATTACTTTACCTGAAGTTCTTAAAGTGTCTCTTAAATGGAATCTAGCTGATTGTATTTCTGCA
This mutation is a 398 bp deletion beginning at Chromosome 3 position 134,861,032 bp and ending after 134,861,429 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44714 | CTT TGT TTG ACA GGA CTT TTC C | Wild type Forward | A | |||
44715 | ACT GTT CTG CAG TCA GAC ATT CG | Common | A | |||
44716 | AAG GGC TGG TAT TTG TAT TGT G | Mutant Forward | A | |||
44717 | Fluorophore-1 | CCC TGG CAA ACC TCA GC | Quencher-1 | WT Probe | ||
44718 | Fluorophore-2 | AAG TTC AAC AAA TTC CAA GTC CTG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44714 | 0.40 uM |
44715 | 0.40 uM |
44716 | 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 |