SSLM cDNA synthesis
(I) First strand synthesis
RNA 1-5 μg
OligodT20 primer (100pmol/ml) 1 μl
DW
Total 23 μl
70°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 1μl
pre incubate at 40°C for 5 min
SuperScriptIII 2 μl
Total 40 μl
40°C for
30sec, 1min, 2min, 3min, 4min, 30min
take 6 μl
Stop the reaction to transfer the aliquate to 100 μl of the 0.3N NaOH, 50°C for 30 min to hydrolysis RNA.
Add 30 μl of 1N HCl
ここからはキアゲンのキット（キアクイックゲルイクストラクションキット、ミニエリュート）で精製する．
Add
QG buffer 500 μl
Apply on the QIAMinElute Spin filter
Wash with NEW solution 800 μl x2 times
Wash with 80% EtOH once
elute DNA by adding 12 μl of EB buffer
Take 9 μl
(II) Oligonucleotide ligation
cDNA 9 μl
RS 30N5 1 μl
Ligation High (TOYOBO) 10 μl
Total 20 μl
12°C for overnight
(III) Purification and PCR amplification
S400 HR (スピンカラムのキットでも可能）RS30N5の除去
Add 30 μl of TE
Apply on S400 HR (TE with 0.05% Tween 20) column
Centrifuge for 1000 rpm for 5 min
Apply with DW 50 μl
Centrifuge for 1500 rpm for 5 min
Total collection 100 μl
1st PCR (ottional)
Linker-ligated ss-DNA 20 μl
x5 PCR buffer 20 μl
Primer (21/31 primers, 100p/μl each) 1 μl
Enzyme 1 μl
DW
Total 100 μl
PCR reaction 55°C 30sec, 72°C for 3min 15-20 cycles
Electrophoresis 5 μl
2nd PCR
x5 PCR buffer 20 μl
Primer (22/32 primers, 100p/μl each) 1 μl
enzyme 1 μl
DW
Total 100 μl
Template (from 1st PCR ) 5 μl
PCR reaction for 15-20 cycles 55°C 30sec, 72°C for 3min
If desired fraction is determined, scale up the 2nd PCR reaction up to 1 ml.
Purify the cDNA by the Centricon-100.
no iframe