SLIC Method cDNA synthesis
(I) First strand synthesis
RNA 5 μg
OligodT primer(100pmol/ml) 1 μl
DW
Total 20 μl
65°C for 5 min , and on ice for 5 min.
x5 first strand buffer 8 μl
0.1M DTT 4 μl
10mM dNTPs 2 μl
RNase inhibitor 2 μl
SuperScriptII 4 μl
Total 40 μl
40°C for 1 hour
RNase A 4 μl
RNase H 4 μl
37°C for 30 min
DNA binding buffer 50 μl
Apply on the QIAprep Spin filter
Wash with NEW solution 800 μl x3 times
Wash with 80% EtOH 800 μl x 2 times
Elute with 100 μl of DW
Electrophoresis 5 μl
Evaporate
(II) Oligonucleotide ligation
x10 buffer 2 μl
RS oligo (RS14, RS13, 5pmol/μl) 4 μl
DW 2 μl
T4 RNA ligase 2 μl
50% (W/V) PEG6000 10 μl
16°C O/N or 37°C for 6 hours
(III) Purification and PCR amplification
DW 30 μl
Apply S400 HR (TE with 0.05% Tween 20) column
Wash with DW 50 μl
PCR
oligo-ligated ss-DNA 20 μl
x5 PCR buffer 20 μl
Primer (LL13P, LL14P, 100p/μl) 1 μl
Enzyme 1 μl
DW
Total 100 μl
PCR reaction for 15 cycles
Electrophoresis 5 μl
PCR
x5 PCR buffer 200 μl
Primer 10 μl
enzyme 7 μl
DW
Total 1 ml
Template 50 μl
PCR reaction for 10 cycles
Purify using Centricon-100 by 3KG
Twice wash with DW 2 ml
15N OligodT Mix (LL13とペアー)
GCTTCTCGTGTTCATCGTTAGCGGCCGCTTTTTTTTTTTTTTTTTTV
16N OligodT Mix (LL14とペアー)
AGAAGAAACAAACCAACCACGCGGCCGCTTTTTTTTTTTTTTTTTTV
RS 13
PGTCGACAACAAGGAGGAACAGAAB
RS 14
PGTCGACGTGGTTGGTTTGTTTCTB
LL13 primer
CTTTTCTGTTCCTCCTTGTT
LL14 primer
AGAAGAAACAAACCAACCAC
LL15 primer (LL13とペアー)
GCT TCT CGT GTT CAT CGT TA (Optimal Annealing Temperature:51.4｡with LL13)
LL16 Primer (LL14とペアー)
AGA AGA AAC AAA CCA ACC AC (Optimal Annealing Temperature:51.5｡with LL14)
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