Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 85 bp
Wild Type = 147 bp
>chr6:113075919-113076065 147bp CTTCCCTCGTGATCTGCAAC AATCTGTGGGAAGTCTTGTCC
Wt Sequence: ctctcccaaagtcgctctgagttgttatcagtaagggagctgcagtggagtaggcggggagaaggccgcacccttctccggaggggggaggggagtgttgcaatacctttctgggagttctctgctgcctcctggcttctgaggaccgccctgggcctgggagaatcccttccccctcttccctcgtgatctgcaactccagtctTTCTagaagatgggcgggagtcttctgggcaggcttaaaggctaacctggtgtgtgggcgttgtcctgcaggggaattgaacaggtgtaaaattggagggacaagacttcccacagattttcggttttgtcgggaagttttttaataggggcaaataaggaaaatgggaggataggtagtcatctggggtttta
Mutant Sequence: GGCCTGGGAGAATCCCTTCCCCCTCTTCCCTCGTGATCTGCAACTCCAGTCTTTCTAGccttaattaagggatctgtagggcgcagtagtccagggtttccttgatgatgtcatacttatcctgtcccttttttttccacagctcgcggttgaggacaaactcttcgcggtctttccagtggggatcgacggtatcgtagagtcgaggccgctctagaactagtggatccggaacccttaatataacttcgtataatgtatgctatacgaagttattaggtccctcgacctgcagcccaagctagcttatcgataccgtcgagatcttgggtacccgctcgaaattcaccatgccaaagagacccagacccTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTAATGAGGTCGG
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
21309 | AAT CTG TGG GAA GTC TTG TCC | Wild type Reverse | A | |||
21310 | Fluorophore-1 | TAA CCT GGT GTG TGG GCG TTG T | Quencher-1 | WT Probe | ||
36158 | CTT CCC TCG TGA TCT GCA AC | Wild type Forward | A | |||
46899 | GGT ACC CGC TCG AAA TTC A | Mutant Forward | A | vector | ||
46900 | GCT CTA ATG CGC TGT TAA TCA CT | Mutant Reverse | A | rTta | ||
46901 | Fluorophore-2 | CAT GCC AAA GAG ACC CAG ACC C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
21309 | 0.40 uM |
36158 | 0.40 uM |
46899 | 0.40 uM |
46900 | 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 |
---|---|
024746 | STOCK Gt(ROSA)26Sortm1.1(rtTA,EGFP)Nagy Hprt1tm1(tetO-Dkk1)Spdl Tg(TCF/Lef1-lacZ)34Efu/J |
031011 | STOCK Gt(ROSA)26Sortm1.1(rtTA,EGFP)Nagy TgTn(pb-tetO-Pou5f1,-Klf4,-Myc,-Sox2,-mCherry)250Nagy Tg(Pou5f1-EGFP)1Nagy/J |
2 strains use this protocol |