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Wierd trouble - dunno where my insert is?? (Sep/14/2009 )

Hi all,
I just got a insert in pcDNA3 vector from another lab ;) but the problem is the other lab has no clue on where the insert is actually located <_< now what am I supposed to do?? Where do I start? I'm thinking instead of cutting out the insert can I just do a PCR for the whole length of the insert and amplify the sequence....and go ahead with my work...I need the insert for site directed mutagenesis....Kindly tell me something.....

:( Alex


Your idea of PCR on the whole length of the insert should work. You'll still want to cut the plasmid, though, if for no other reason than the PCR will be easier if you're not using circular DNA. Look up the plasmid map, see what cuts it, and cut it with an enzyme that won't cut your insert (I'm guessing you know the whole sequence of your insert).


-Carlton H-

Maybe you can try to design primers that bind near the end of the insert and try to sequence out from the insert. If you have the sequence of pcDNA backbone you can deduce where it is inserted.


Do you have the sequence of the insert? If so, look for unique cut sites. Find unique sites in the vector and do a digestion. The fragment sizes will tell you where the insert is in the plasmid.