TA cloning nightmare

[Amna Saeed]

I am having a lot of trouble trying to subclone my PCR products 
(amplified using Clonetech's Advantage Klentaq) into my expression 
vector (Promega's pGL3 Luciferase Reporter vector) I have tried 
everything from blunt ending my vector, adding T overhang, 
dephosphorylating, adding A overhang to my product, phosphorylating, 
etc. I cannot use sticky-end cloning because all the restriction 
enzymes present in the polylinker also chop up my product into 
multiple fragments. Also I have found that using the protocol from 
Biotechniques for adding T's to EcoRV cut pBluescript vector does not 
seem to work in adding T's to my SmaI cut vector. I did a test 
ligation and after ON incubation at 15oC, all the supposedly T-ed 
vector had recircularized, i.e it had no T's to begin with ??
	Does anyone have any experience in working with this vector or any 
alternative ideas as to how I could get my piece in (TA cloning 
related or otherwise)? Interestingly, the one clone that I did manage 
to find after screening 300+ colonies (this vector has no antibiotic 
or blue-white selection) should have contained my PCR product of 
approx. 4.5 kb but after sequencing and nested PCR I discovered it had 
somehow been truncated (during subcloning) to 3.7 kb. Any ideas as to 
what might have caused this bizarre phenomenon??
																																															In desperation,
																																															Amna Saeed
																																															Dept. of Biology
																																															Northeastern Univ.
																																																asaeed at neu.edu