phyA dCAPS F (EcoNI cuts WT) |
TAACTGAATACACCATTCCCTTAACC |
47⋅C/45 s |
phyA dCAPS R (EcoNI cuts WT) |
ATAATCGCTCTATAGTCACC |
WT 215/20; phyA 235 |
phyB1 dCAPS F (HinF1 cuts WT) |
CTAAAATTCAAAGAGGAGGTCAGATT |
58⋅C/20 s |
phyB1 dCAPS R (HinF1 cuts WT) |
GAAGGGGTAAAAAGGGTCCTAA |
WT 172/20; phyB1 192 |
phyB2 F WT specific |
CCCTTTTTCCTTTTCTGACC |
62⋅C/45 s |
phyB2 R non-specific |
GACAATATTGAGGATGGGTA |
WT 513; phyB2 absent |
phyB2 F mutant specific |
GTCTTGATTTCGTCTGGA |
66.5⋅C/45 s |
phyB2 R non-specific |
GACAATATTGAGGACGGGAGAGTT |
WT absent; phyB2 452 |
phyE amiRNA F |
GCTCGGACGCATATTACACA |
58⋅C/30 s |
phyE amiRNA R |
ACCATGATTACGCCAAGCTC |
WT absent; PHYE amiRNA 493 |
positive control F |
TGATGTTGATGGGCAGGTTA |
58–67⋅C/30 s |
positive control R |
CACTCAGAACACCAGCCAAA |
676 |
Genotyping primers for phyA, phyB1, and phyB2 (Weller et al., 2000). |
PHYE
amiRNA generation primers
|
I miR-s |
GATAAATCTGACAGAAGACGCTGTCTCTCTTTTGTATTCC |
II miR-a |
GACAGCGTCTTCTGTCAGATTTATCAAAGAGAATCAATGA |
III miR*s |
GACAACGTCTTCTGTGAGATTTTTCACAGGTCGTGATATG |
IV miR*a |
GAAAAATCTCACAGAAGACGTTGTCTACATATATATTCCT |
Oligo A |
CTGCAAGGCGATTAAGTTGGGTAAC |
Oligo B |
GCGGATAACAATTTCACACAGGAAACAG |
PCR conditions: http://wmd3.weigelworld.org/downloads/Cloning_of_artificial_microRNAs.pdf
|
qPCR primers |
PHYB1 F |
CAATGCTCTAAGAGGCGTGGA |
61⋅C/1 min |
PHYB1 R |
CTGGAGCAAGCATTAACCACCA |
2-step protocol, 40 cycles |
PHYB2 F |
GGAAGGGTGGGTAGAAGTCC |
61⋅C/1 min |
PHYB2 R |
GGGCAAACAATCCTGAACTC |
2-step protocol, 40 cycles |
PHYE F |
GGAGACAAGTGAAGCCTGTGAG |
60⋅C/1 min |
PHYE R |
TGCCGTCCTCTATACCTCCAA |
2-step protocol, 40 cycles |
control F (Solyc03g111090) |
CTGACTTCTCAGCAGAACTCCAAT |
60–62⋅C/1 min |
control R (Solyc03g111090) |
TTCACCCTTTTCAATGCTCTTCTC |
2-step protocol, 40 cycles |