Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 99 bp
Wild Type = 140 bp
>chr3:146896888+146897027 140bp TAAGCCCGTGTCATTTCCA CTTTGTCCACTAAGACAATCCATTC
Sequence (deleted region in lower case)
TGTTGGGGAAGCCCTGAGGGTGAATGTACCTGTGTAGAGTTATATCAGCTGTCTCTGTGTTTTTACTGTACTAAAGTCACTCTCTAAACCctctgCAAGTGCTGTGTTTTGGGAAATTGCGCAGGACTATGACTAAGGCTTTTTGGTGCAACCTAATTCAGTTCCCAGCTTCTGTGGAGAGCAATTTGGTAAAAGTGCCGTCACTGGTAATGAGCTTATGCTATAAGGAGTGGggccagtctgcacctggaacactgtgggtatacgcatgtgcatttccattgtgaatgtctttaaggtgttctggctcaggtttgatgtaatcgtctcttttcagttcgtttagtaatagatgagatgggatttatgaaaactcctgatgaagatgagacaagcaatcttatctggtgtgatgctgctgttcagcaggagaagattacagacctgcagaactaccaggtggggactgacagtcagcatcggggagaaagtcacaccacttccaccacttccgtcatggttttatttagcttcttaatctgtccacatattagctttcagttctgagttccctgggccgcctgtctcgttgctcgctggctgtgctgactctttgcattaagcccgtgtcatttccaaactcaacctttagcttttttctttaaagatggatcaacagggaaaaattctaagttgatgctttgagttatgtacaccttaagatgagtgacagGGAATGGATTGTCTTAGTGGACAAAGACCTTATAGCTTCAGCTCCATTAGTCTGACTTTTACATCTATAGTGCAGTACAAATGTGTTTAGAAGAAATGGAAAGCAAATTGAAAAACCTACAAGGGATCCAAAGCAATCGATGTTTGCTACTTACCCTCCCTCCCTTCTTCCCCTTCCTCTCTCCTCCTCCCTCCCTCCCTCTCTCCTTCCCTCCTTTCCTCCCTCCCTTCCTCCCTCCTTCTCTCCTTCCTTCCCTCCCCTTTCCTTCCTCTCCCCTCCTTCCC
500 bp deletion beginning at Chromosome 3 positive strand position 146,896,502 bp and ending after 146,897,001 bp (GRCm38/mm10). In addition there is a 5 bp deletion (CTCTG) 138 bp before the 500 bp deletion
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37693 | GTT CCC AGC TTC TGT GGA GA | Mutant Forward | A | |||
37694 | CTT TGT CCA CTA AGA CAA TCC ATT C | Common | A | |||
37695 | TAA GCC CGT GTC ATT TCC A | Wild type Forward | A | |||
37696 | Fluorophore-1 | AGT GCC GTC ACT GGT AAT GA | Quencher-1 | MUT Probe | ||
37697 | Fluorophore-2 | TGG ATC AAC AGG GAA AAA TTC T | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37693 | 0.40 uM |
37694 | 0.40 uM |
37695 | 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 |