Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.
Mutant= 145 bp
Wild Type = 63 bp
Wt Sequence:tccatggattgctgtattggaatcaaacagaaatctatgtcattcacagcagtaacctcctgggaatacttcaagagacggagaaagggcgactgactgtgccctccgtcctttttcgcaatcctttattCTGTTCGAAtcataatttgt
tttcctgagacagggtttctctgtgtagtcctggctgtcctgggactcccctctgtagaccaggctggcctccaactgtctctgcctcctatgtgctgggatcaaaggtgcctgccaccaccgcctggcttcttagagttgg
Mutant Sequence:GAGACGGAGAAAGGGCGACTGACTGTGCCCTCCGTCCTTTTTCGCAATCCTTTATTCTGTTCGAtaagcttgatatcgaattcataacttcgtataatgtatgctatacgaagttatctgcagcccg
ggggatctgatatcaTCGAATCATAATTTGTTTTCCTGAGACAGGGTTTCTCTGTGTAGTCCTGGCTGTCCTGGGACTCCCCTCTGTAGACCAG
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
31852 | CGT CCT TTT TCG CAA TCC T | Forward | A | |||
31853 | GAA ACC CTG TCT CAG GAA AAC | Reverse | A | |||
31854 | Fluorophore-1 | CAA TCC TTT ATT CTG TTC GAA TCA T | Quencher-1 | WT Probe | ||
31855 | Fluorophore-2 | TGC TAT ACG AAG TTA TCT GCA GCC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
31852 | 0.40 uM |
31853 | 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 |
Stock Number | Strain Name |
---|---|
026512 | B6(Cg)-Tg(TPO-cre/ERT2)1139Tyj/J |
032435 | B6.129-Krastm4Tyj Trp53tm1Brn/J |
037954 | B6.Cg-Gt(ROSA)26Sortm4(CAG-cas9*/GAG-POL,-mNeonGreen)Tyj Trp53tm1Brn/TyjJ |
033754 | STOCK Brca2tm1Brn Gt(ROSA)26Sortm3(CAG-EYFP)Hze Trp53tm1Brn Nkx3-1tm4(cre/ERT2)Mms Ptentm1Hwu/AbshnJ |
033755 | STOCK Gt(ROSA)26Sortm3(CAG-EYFP)Hze Trp53tm1Brn Nkx3-1tm4(cre/ERT2)Mms Ptentm1Hwu/AbshnJ |
033756 | STOCK Gt(ROSA)26Sortm3(CAG-EYFP)Hze Trp53tm1Brn/Trp53tm3Tyj Nkx3-1tm4(cre/ERT2)Mms Ptentm1Hwu/AbshnJ |
032429 | STOCK Krastm4Tyj Trp53tm1Brn Tg(Pdx1-cre/Esr1*)#Dam/J |
7 strains use this protocol |