help with ligation

Edgar Valencia edgar at cifn.unam.mx
Wed Apr 10 16:00:21 EST 1996


Hi Victor:
There are several things that can affect the ligation. One are the salts,
we prefer to precipitate DNA with EtOH and Ammonium acetate and then do
the washes with etOH 70% and resuspend in H2O or TE 10/1 not TE 50/20
We also extract the DNA after digestion with phenol-chloroform-isoamilic
alcohol and chloroform-isoamilic as in Alkaline lysis or using columns
Sometimes if we abuse with the alkaline phosphatase cant do the ligations
Those are the points we take care of.
Finally, which size are your inserts and your vector? if your bands are 
small, try using higher ratios than 3:1
I hope this helps

                          Edgar Valencia Morales
                               CIFN - UNAM
                                  MEXICO
                        e-mail edgar at cifn.unam.mx





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