Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr10:88822194-88822288 95bp ACAGCCAAAGCAGCAAGACT TTGAGAAGAGGGGAGAACCA
Mut= 81 bp
Wt= 95 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AATGCAGAACTGAAGAGTCCTACATTTTTTCTCTGCAGGCAGTTTTTCCTGTGACTATTACTTGGGCTTTAAACAGTTATTTGCTACAGCCAAAGCAGCAAGACTTGTTTTTGTAATTCACCTGctgctggtagacctgctgctggtagactgaggaagttggttctcccctcttctcaaaaacacaaaaagggggtttttaaccccttatccttgtcctaagatgctggctgtggtccgtgctctgcagcttggtcagttgtcctggagttgcctcactaacagctgccttgctcttggcaggacgtcgagacttacttcttcgaggctctgctgaagtggagagagctgaaccttacggaacatttcggtaccgtctgcttctttctacttcatcttcccataatttgcacatcttatgttcccaacaactagtgaggcgatggagaaatagaacataatcctttttgctttctttcttttttccttttgttaatccagggaaattttacaaagaagtcattgacaaatgtcagtcttttaaccagttggtctatcatcaaaatgaaattgtccagagcttgaagactcacctgcaaattaggaacagtttggcctaccaacctcttttggagtaagtcacatctcagatgaaggctcaatagtgtttatctgagttctaaggaaaaccatgcttcatcactggccttaaattacggtctgtaatttagaagcaggatgagtatgccttccctgttagtttactgtatcagaaaaagatgtgtttctgtaaagcagcatcctggtgtacattgCACTTTCTCTGTGCTCCTAAGAGAGCTGTATGTTTCCTTGAAACCAGGGATGGGTGGCCCCAGTTCTGCTATCACTGTTTCTGACTTGAGACCAGGGATGGGTGG
This mutation is a 691 bp deletion beginning at Chromosome 10 position 88,821,559 bp and ending after 88,822,249 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
41179 | ACA GCC AAA GCA GCA AGA CT | Common | A | |||
41180 | TTG AGA AGA GGG GAG AAC CA | Wild type Reverse | A | |||
41181 | TTC AAG GAA ACA TAC AGC TCT CTT AGG | Mutant Reverse | A | |||
41182 | Fluorophore-1 | CTG CTG CTG GTA GAC TGA GGA | Quencher-1 | WT Probe | ||
41183 | Fluorophore-2 | CAC CTG CAC TTT CTC TGT GC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
41179 | 0.40 uM |
41180 | 0.40 uM |
41181 | 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 |