Cloning of PCR product

[vinayaphatak]

Hi,

I am trying to clone my PCR product into pEGFP-N1 plasmid. My PCR product is 349bp. My primers have sites for BamH1 and HINDIII along with 4 more random bases. Double restriction digestion was done with HINDIII and BamHI from promega. I did tried ligation for 3 hours at RT with Promega and Overnight too. I get more colonies in my plates with insert than the control plate, but after screening the colonies I get them without my insert. I was using Taq polymerase for my PCR so thought may be due to A overhangs my restriction digestion is not working so I tried with phusion too. I also thought and tried the Zero Blunt TOPO kit from invitrogen to clone my product but unfortuantely failed too. Please give me suggestions for where I am going wrong.

[Warthaug]

It sounds like one of your restriction enzymes may not be working. Try digesting your plasmid with each enzyme individually, and then run a diagnostic gel to see if this linearizes the plasmid (you'll no longer see the supercoiled form if they are). If both are working, maybe try doing individual digests (i.e. digest with HindIII, purify, then with BamHI).

Another possibility is that you are using way too much DNA. Try diluting everything 1:100, but otherwise keep everything else the same.

Bryan