Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr10:56196823-56196927 105bp AAGTGTTTGGTCTGTGCTGCT GGTGAGAGGGTAAAAGTCTGTG
Mut= 107 bp
Wt= 105 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case; and insertion with carrots ^g^ (T insertion):
TCAAAATGTAGTGTTGCCTACTGTGCCACTGTTGGATGGCACCATTAAAGCCTCCAGCACCTCTGTGCAAGCCTGCAGCTTCATTTCACAGCACCACGTGACCCTTCCTCCCGCCACCATTCACTCCTACTTCCTGCTGTGACCAGATTTCACAGCAGTGCATTTTGTTGTTGGTTTTACCCTTAGTTGTGGATAATTTTACTTTTGGCTAAATGAAGACATGCCCTTTAGAATAGACACGGCCTTCCCCTGG^tttctgcgt^AGTGTATCTTGTGTTTATTTTCTGTTCCATGCGAGAAGTGTTTGGTCTGTGCTGCTGTAAGATCGCAAATGAGGATGGGCTGTTttgcttccctaggaccggattttaaagttctacgcacagacttttaccctctcaccgttacatatgaccaaggagatttatacaagcttaggtatgttcaccaaatgacttcaattgttgttctcctatatgcacttaaaatcccgtaagaaatgacttcttccagaagcattgcgtaatatatataacgactgtatattttccctttccttttcagctaaatatctggaggtatattttctttctagagaaaatcaccttcctactctttcaactggagtagacataaccagtcccaatgtgacccgcttactgaaaaaggtatccacttactatgatgtactgtatggaaagtagggctatatttaagaattaaactcttttgagagcttttgtatactgcttataggttcgccttctgaatgagtaccagaaagaggctccatctttttggattcgtcacccagagaagtaagttccccaactcctgtgatgtaaggtataggctatgtccttcaattatttcaaggacagaaggaacagtagtcttgagcgatGGACATGGTTCTATATGCCACTTAGTTTTTGTCAGCTTGACACCAAATAGGACCACCTAGGAAATAGACCCTCATCTGAGGAACTGCCCTCATCACTTGCCTATGTGTGTGTTTTTGTAACTGTAAATTGATATAGGGGGCCTCCCAGGGGCAATACCTTCTGTAGGCACATGGCCCTGGGCTGTATAAGAAAAGTAGCAAGCTTGAGGAGGCAAGACAGCAAGCAACGCCTCAATGGTTTTTGCCCCAAATTCCTGTCTTGAGCTCCTGGTCTGGCTTCCCTT
This mutation is a 557 bp deletion beginning at Chromosome 10 position 56,196,322 bp and ending after 56,196,878 bp (GRCm38/mm10). In addition, there is a 1 bp (T) insertion and a 9 bp deletion (ACGCAGAAA) 84 bp after the deletion.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48684 | AAG TGT TTG GTC TGT GCT GCT | Common | A | |||
48685 | GGT GAG AGG GTA AAA GTC TGT G | Wild type Reverse | A | |||
48686 | AGG TGG TCC TAT TTG GTG TCA | Mutant Reverse | A | |||
48687 | Fluorophore-1 | CTT CCC TAG GAC CGG ATT TTA A | Quencher-1 | WT Probe | ||
48688 | Fluorophore-2 | ATG GGC TGT TGG ACA TGG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48684 | 0.40 uM |
48685 | 0.40 uM |
48686 | 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 |