Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 63bp
Wild Type = 124 bp
>chr14:51106133+51106256 124bp CTGTATGTTGAGCCATCTGT AATGGCCATTTGTATTTGAC
Wt Sequence (deletion in lower case):
TTTTCTTACAAAACCCAAATTCCAGTTTTATGTAGATTTATCTTTCCTTGTTTTATACTCCAGTCAGGCAGGCCTGGGCAACTGACCACCCAGTCATGTTTGTTCTGACTTGTTCTGTTAGCTGTACGCCTGTCAATTAGTAAAGAGAACAGTGTTTGCTTTCAAGGAAGGCCATGTTGACAAACTAGCCACTCTCAGAGaaaggggtacaaaagggaagagcaggaagaagggggagatcttaagcagaaggaggggcaagagagggttctaagagtttagtttgcttttctcattacctctccagagcccagtccttaccatcctttctccttgttttcttctcaggcactttctaggtaatgatggatctacagaggactcagtccttgcttctgctcttggtgctgaccctgctggggttagggcttgtacagccctcctatggccaggatcgaatgtaccaacggttccttcgacagcatgtggaccctcaggtgacaggtggcaatgacaactactgcaacgtgatgatgcagagacggaagatgacttctgtccagtgcaaacgcttcaacaccttcatccacgaagacatctggaacattcgtggcatctgcagtaccaccaatatcctgtgcaagaacggccagatgaactgtcacgaaggtgtagtgaaggtcacggactgcagagagacagggaactccaaggcccccaactgtagatacagggcaagaaccagcactaggcgagttgtcattgcctgtgagggtgacccagaggtcccagtgcactttgacagatagatgctgtcctgcagctgttacagctgatagttgacctctgagtcagtgagaccaacaatgagtggtggccccaggctttgtttcctttgctcgtgccttatactcacatatatccaatataacccatggtgttaagcgcatcccatccaagggaagaaagaacccactgctcacagcactgatggcttggttctcccagattctatcccctggaacttatgcattacatgtattcagatagtatctcaacgattatagagagccttttctgtccaagctgtctcattccagtcctctaacaccccttgagaccaattctgccactgtatttagaagctttgagttaaaggatttgacgaccacaaaaaaccaaaaccaaaaccaaaaacaaaaacccagtggaagatctcagacaaaaatccaactggacgggctactcacaaaaggtgttacccacttcgttatatatactttctgttattccaaaatgtcatcattagggaagccactaagagtttggaaataggggctggagagatgcctctgtggttaagagcattggctgctcctctagaggacccaggttcaattcccagcgcccacacatcagctcacaactgtctgtaacccagttctaggtgatttgataccctcacacagacatacatgtagacagatcaccaatgcatataaaattaaaataaagattttggaaacaacatattgccaaaatgagaattacttggtattattttggtctgtggcttctaagattttttaaaatacatgaaaaatgtagtaaatcactaaaattcatttatccttctcctttgaaagattttcaatattttagtataataaagagctgtatgttgagccatctgtgtcgggatttgtgttctcctgttctttttcagttcctctctagtctatgaactctcatgtcccttagtgtccctatACATTCTGGTCAAATACAAATGGCCATTAGATCCATCATGTGACATCTGACTTTGTCTCCTTTACTCCACTGAAAGTGATCCCTCTCATACACAAGACCTGAAATAAGAACTCAGAAGTGGCCTTTGCTAAAAAGCACCAATTCTTTATGCAACAAATTGAGTGTGTTGCATGATTAAGC
This mutation is a 1570 bp deletion beginning at Chromosome 14 position 51,104,659 bp and ending after 51,106,228 bp (GRCm38/mm10)
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
68976 | CTG TAT GTT GAG CCA TCT GT | Wild type Forward | A | |||
68977 | AAT GGC CAT TTG TAT TTG AC | Common | A | |||
68978 | GGA AGG CCA TGT TGA CAA A | Mutant Forward | A | |||
68979 | Fluorophore-1 | CAT GTC CCT TAG TGT CCC TA | Quencher-1 | WT Probe | ||
68980 | Fluorophore-2 | CTA GCC ACT CTC AGA GAC ATT CT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
68976 | 0.40 uM |
68977 | 0.40 uM |
68978 | 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 |