41. Random hexamer primers (100 μM stock).
42. Klenow fragment enzyme.
43. dATP/dGTP/dTTP mix (1 mM each, without dCTP)
prepared using a set of individual dNTP solutions.
44. [α
32 P]-dCTP with a specific activity of 6000 Ci/mmol and
radioactive concentration of 10 mCi/mL (see Note 3).
45. T4 polynucleotide kinase (PNK).
46. [γ
32 P]-ATP with a specific activity of 6000 Ci/mmol and
radioactive concentration of 10 mCi/mL (see Note 3).
47. Illustra MicroSpin G-25 columns (GE Healthcare) for probe
purification.
48. Wash buffer: 2Â SSC + 0.5% sodium dodecyl sulfate (SDS).
49. Stripping buffer: 0.1% SDS.
50. Microwave.
51. Phosphor-imager (e.g., Typhoon FLA-9500; GE Healthcare).
2.4 DNA Oligonucleotides for
Hybridization Probes
Organism
Name
Sequence
Size
(nt)
Hybridization/
Wash temp.
DNA primers used to make Klenow probes
B. distachyon
Bd_LTR_F01 GATGGACCTAACACATT
ACGAGAG
24
PCR using F01 and R01,
then Klenow labeling:
35/37
C, or
42/45
C
B. distachyon
Bd_LTR_R01 GTTCTTCAAATATTGTC
TGTTGGAG
25
DNA oligos used as 5
0 -end-labeled probes
B. distachyon
Bd_LTR
AGTGGCTCTGCCGCCCG
GAAGCTT
24
35/37
C
A. thaliana
LTR_META1 GCCCATCATCTAAGCCCA
TCATCT
24
40/40
C
A. thaliana
SIMPLEHAT2 TGGGTTACCCATTTTGAC
ACCCCTA
25
35/37
C
A. thaliana
AtREP2
GCGGGACGGGTTTGGC
AGGACGTTACTTAAT
31
35/37
C
B. distachyon
or A. thaliana
(Universal probe)
miR160
TGGCATACAGGGAGCCA
GGCA
21
35/37
C
Genome-Scale and Northern Blot Analyses of siRNAs
395
42. Klenow fragment enzyme.
43. dATP/dGTP/dTTP mix (1 mM each, without dCTP)
prepared using a set of individual dNTP solutions.
44. [α
32 P]-dCTP with a specific activity of 6000 Ci/mmol and
radioactive concentration of 10 mCi/mL (see Note 3).
45. T4 polynucleotide kinase (PNK).
46. [γ
32 P]-ATP with a specific activity of 6000 Ci/mmol and
radioactive concentration of 10 mCi/mL (see Note 3).
47. Illustra MicroSpin G-25 columns (GE Healthcare) for probe
purification.
48. Wash buffer: 2Â SSC + 0.5% sodium dodecyl sulfate (SDS).
49. Stripping buffer: 0.1% SDS.
50. Microwave.
51. Phosphor-imager (e.g., Typhoon FLA-9500; GE Healthcare).
2.4 DNA Oligonucleotides for
Hybridization Probes
Organism
Name
Sequence
Size
(nt)
Hybridization/
Wash temp.
DNA primers used to make Klenow probes
B. distachyon
Bd_LTR_F01 GATGGACCTAACACATT
ACGAGAG
24
PCR using F01 and R01,
then Klenow labeling:
35/37
C, or
42/45
C
B. distachyon
Bd_LTR_R01 GTTCTTCAAATATTGTC
TGTTGGAG
25
DNA oligos used as 5
0 -end-labeled probes
B. distachyon
Bd_LTR
AGTGGCTCTGCCGCCCG
GAAGCTT
24
35/37
C
A. thaliana
LTR_META1 GCCCATCATCTAAGCCCA
TCATCT
24
40/40
C
A. thaliana
SIMPLEHAT2 TGGGTTACCCATTTTGAC
ACCCCTA
25
35/37
C
A. thaliana
AtREP2
GCGGGACGGGTTTGGC
AGGACGTTACTTAAT
31
35/37
C
B. distachyon
or A. thaliana
(Universal probe)
miR160
TGGCATACAGGGAGCCA
GGCA
21
35/37
C
Genome-Scale and Northern Blot Analyses of siRNAs
395