help: cloning oligos

Soon Paik paiks at ari.net
Sun Jul 2 00:13:45 EST 1995


Dear Netters,

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 
following.
5'-aattcc----------------------tc-3'
  3'-gg----------------------agagct-5'
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?
Any suggestions?

Soon Paik, M.D.
Lombardi Cancer Center
e-mail: paiks at gunet.georgetown.edu





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