Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 108 bp
Wild Type = 118 bp
>chr2:38950922+38951039 118bp GACAGGAGATGGGAATGAAT AACGTTGTTCCCAGAGTAAA
Wt Sequence (deletion in lower case):
CCCATAAACCAAGACCAGTGAGCGAGAGACAAGGCTAGGAGGTCTTCCAAAGAGCCAACAAAACAGGTGGACAGGAGATGGGAATGAATATGTAACCTACCggattgggtctcaagcctgcagagaaaatctgtatggagaaacagaatccaacaactatggatgattttactctgggaacaacgttacagacactgcgactacctccagctctcacacactgagctactgtgactggcagcccctcattttactgtcacccaaatctgacacttctattcttcccaggacacagctggagggcaaggcagagatctcaacaaatatggcagcatacagaagagcttctcagacaagggcctggcaaagcctcccaaggagaagctgctgaaagtggagaagctgaggaaggagagcatcaagggcagaataccccagcccacagccaggccacgtgcgctggctcagcagcaggcagtgatccgaggcttcacctactacaaggcaggcagacaggaggccagacaggaggccagacaggaggcacccaaggctgccgctggtgagtggaagggagccgggcctggttgacggatggctgcttccagagatccacctgccttcgaagcagccacaaagtcacagatctccgcatcactgtttagttttctagaatcctttgctcaaccatacACTGGGAAGAGGCCCACAGGCAGGGTTGCTAATGGGGAGAGAATAGAGGCCTCTCTTTCAGTACTTCAAGCCTCACCTTTTCTGCACTTCCTTTTATTCAACGACCTGCTGCAGGTCAGCTACAGACCAAGTGAC
This mutation is a 592 bp deletion of region Chr2:38,950,954-38,951,545 (GRCm38/mm10)
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
70979 | GAC AGG AGA TGG GAA TGA AT | Common | A | |||
70980 | AAC GTT GTT CCC AGA GTA AA | Wild type Reverse | A | |||
70981 | GTG AGG CTT GAA GTA CTG AA | Mutant Reverse | A | |||
70982 | Fluorophore-1 | TCT CAA GCC TGC AGA GAA A | Quencher-1 | WT Probe | ||
70983 | Fluorophore-2 | ACC TAC CAC TGG GAA GAG GC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
70979 | 0.40 uM |
70980 | 0.40 uM |
70981 | 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 |