SOS!--PCR restriction cut

Edgar Valencia edgar at cifn.unam.mx
Mon Mar 18 12:01:18 EST 1996


Hi Wenjin Yu:

Just a stupid question, but it happened to me. Are your restriction sites
correctly oriented? I mean In your oligos EcoRI is 5' XXXXXXGAATTCxxxxxx 3'?
I constructed some oligos with a "EcoRI site", added the bases at the 5'
end to avoid cutting problems. The PCR was nice, the product has the 
internal restriction sites it should had, but when i tried to cut with 
EcoRI an ligate it it was impossible!. And then i saterted to modify all 
my conditions, from vector:band rates till cleaning the PCR product and 
vector and nothing! and then, just for curiosity I saw the restriction 
table in a catalog and theres a enzyme, AfllI which has the recognition 
sequence 5' CTTAAG 3', the opposite to EcoRI, so I can figure out now 
what happened and ill try with new oligos with the EcoRI site in the rigth
orientation.
At the beggining I though Is stupid! because the restriction sites are 
palindromic sequences, but after check it again, you can see its not the same
I hope this helps

PS By the way
If anybody can tell me the recognition mechanism of EcoRI ill be glad

                          Edgar Valencia Morales
                    Departamento de Genetica Molecular
                               CIFN - UNAM
                                  MEXICO
                        e-mail edgar at cifn.unam.mx







More information about the Methods mailing list