Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:105396607-105396694 88bp AAAAATGCTTCAAACTCAGAAGA TAAACGGAATCAATCCCAAAATG
Mut= 105 bp
Wt= 88 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AAACAAAAAAAAAAAAAAAACAAAAACAAAAACAAACAAAAAAAAAAAAACAAAAACAAAAAAAACCAAAAAACTCCATAGATTTTTTTTCCAAGGAAAATATCCTGTTATTTGATATAAAATTAGAAGAAAAAAAATGCTTCAAACTCAGAAGAAAAAAAATCattgagaattttaattattaccttgccattttacattttgggattgattccgtttattttaaaataccgtcccaaagcaagtcacatgctattttttctgagtattcagtgtcaaaacaaaacaaaacaaagttggttatgaggttttgatgttgaaggcagcactcttctctgtgggacagaccctagataacctgaaggaagggagacaccacctccgtgtgcggcctgccgacattcctgtggctgagagagcatccctgaactactggcggacatggaagtgtatcagcaagccctcggattttccaataaaaagccccgccttcacaggtaccgtcacgggcaggcccacaccctgaagtcaccagcttcgactctcctcgcccgaacagacgttaacctccatctgaactacttctgggtgctgcctctcttttcctcttctcttttgacccagtactgtcctctgcatggctgcttgcttactgggactcagcaccctgtgccattccgtcctgctgtatctaggcttcctttaggtgatatgatgtcatccAGTCTCTCCTGGCTCCATCATTATTTCAACTTACCTCAGACCACTACACAAAAATGCTCAGTGGTCTCTGGCTTCTGAGGCTCTTGCAGCATAGGGACATAAGTCCCTATGCTGGGACATCAGGAG
This mutation is a 559 bp deletion beginning at Chromosome 8 position 105,396,104 bp and ending after 105,396,662 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
42632 | AAA AAT GCT TCA AAC TCA GAA GA | Common | A | |||
42633 | TAA ACG GAA TCA ATC CCA AAA TG | Wild type Reverse | A | |||
42634 | AGC CAG AGA CCA CTG AGC AT | Mutant Reverse | A | |||
42635 | Fluorophore-1 | ATT GAG AAT TTT AAT TAT TAC CTT GCC A | Quencher-1 | WT Probe | ||
42636 | Fluorophore-2 | ATC AGT CTC TCC TGG CTC CA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
42632 | 0.40 uM |
42633 | 0.40 uM |
42634 | 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 |