help: cloning oligos
paiks at ari.net
Sun Jul 2 00:13:45 EST 1995
In order to generate a ribozyme expression construct,
1) I have generated two complemetary oligos (49mers) with EcoRI and XhoI
overhangs on each end. So that when hybridized they will be like the
2) oligos were 5'-kinased in seperate reactions for each using T4
kinase 37 degrees for 1 hour. Enzyme was inactivated by heating for 10
min at 65 degrees.
3) 50 pmols of oligos were then annealed in 10mM Tris, 1mM EDTA, 50mM
NaCl by first heating them for 5 minutes at 70 degree water bath, then
slowly cool to room temp for about 3 hours.
4)vector was cut with EcoRI and XhoI and dephosphorylated with CIAP,
phenol extracted and ethanol precipitated.
5) 0.5 pmol vector and 5 pmol annealed oligos were ligated in 8
microliter volume with T4 ligase.
6) checked many colonies and cannot find the right inserts.
What have I done wrong?
Soon Paik, M.D.
Lombardi Cancer Center
e-mail: paiks at gunet.georgetown.edu
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