(I) First strand synthesis
RNA 5 μg
OligodT primer (100pmol/ml) 1 μl
DW
Total 22 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 1 μl
SuperScriptIII 3 μl
Total 40 μl
40°C for 1 hour
on ice
(II) Second strand synthesis
DW 207 μl
5x 2nd strand buffer 66 μl
20 mM dNTPs 3.3 μl
E. coli Ligase 2.2 μl
Pol I 8.8 μl
RNaseH 2.2 μl
16°C 2 hours
20 mM dNTPs 5 μl
pfu polymerase 4 μl
RNase A 5 μl
72°C for 30 min and 37°C for 10 min
(III) Purification
phenol/chloroform extraction
Centricon-100
EtOH precipitation with glicogen
Or
(III) Purification
DNA binding buffer 350 μl
Apply on the QIApress 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 or EtOH precipitation
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