I've been trying for months now to produce 3 separate clones using the pENTR/D-TOPO directional cloning kit from invitrogen with absolutely no success. I would any and all input on the subject. Here we go
1. PCR using Pfu polymerase fragment (1kb - 1.5kb) using double checked primers with cacc- immediately before the ATG
2. gel extract and purify (promega wizard kit)
3. have followed this with ethanol precipitation
4. measured A260 and calculated DNA concentration of PCR product to calculate 1:1 molar ration vector to insert
5. performed TOPO cloning protocol and transformation (using both Mach1 and Top10 cells - very gently)
6. here's the tricky party - get HUNDREDS of colonies
7. Culture, miniprep and digest with Not1 (which doesn't cut any of my inserts) (used a cut empty vector as comparison)
8. - NO INSERT!!!! all bands are the same size as the cut entry vector (although the last one I did had all bands at 1.5kb and the cut empty vector at 2580kb - go figure)
about to attempt pCR8/GW/TOPO upon receipt of new primers but this is really doing my head in.
I have my heart set on the TOPO system for ease of use of downstream projects i.e. expression of gene in mammalian and bacterial cells, protein purification, site-directed mutagenesis etc. so I REALLY want this to work.
If anyone had any hints or tips I'd be most appreciative!