Zero Blunt cloning - ligation problem? (Jun/26/2008 )
I am trying to clone blunt-end DNA fragments of various sizes (.5-1.5kb) into Zero Blunt ~ 3.5kb in length. These are 10 clone with kb + ladder. Used M13 F and R and ran a PCR and this is the gel pic. Why are most of my inserts the same size? Are these inserts at all? [attachment=4901:06.25.08.1.gif]
I presume you're referring to the fuzzy bands at the bottom. I don't think your PCR has worked at all, and the fuzzy bands are primers. Does the PCR work usually?
Next time you do it, try running just plasmid backbone and seeing what you get.
I think the primers and dntps are at the bottom of the gel pic. What are the two bands I get in each row?
I think (quit sure) these bands are just the coiling vector and there are no iserts or u have problem with the pcr primers or pcr protocol
u have to load ur original vector into the gel with these samples and please send it or loaded here for me