restriction digestion - (Sep/23/2005 )
well i am anew member asking for somthing may sounds silly well i am trying to digest a pcr product and clone it inside pET28a but every time after ligation either no colony is avilable or only few colony with only the vector ..
1 is there any problem of digestion of insert i.e. the PCR product ?
2 is there any chance of self ligation?
more details, please?
what restriction enzymes do you use? other reagents? what is your ligation and transformation protocol?
Yes -- we need further details about what enzymes you used to cut your vector and insert, how big your insert is, how you purified you linerized vector, what ligation method you're using, how competent are your cells, etc.
If your restriction sites are engineered into the 5' end of the primers, how many extra 5' bases did you leave?