RF-Cloning.org Forum

Hello Mr. Steve Bond,
I am completely new to this cloning and hope this beginner question isn't too dense on my part. My RF cloning yields me colonies (2-10ish) after transforming my 2'PCR product into DH5alpha cells, but when I screen my colonies, none of them ever contain the insert and are usually only just the vector sequence.

I believe my 1'PCR is fine as I get enough product after gel extraction of my 1'PCR product. I am currently doing the 3-step 2'PCR at 53C and 55C annealing temperature. I did a negative control (megaprimer not added) which gave no colonies as well as a positive control w/o DpnI treatment which gave 50+ colonies.

I'm planning to try the 2-step PCR protocol on your website next as well as follow the insert:vector ratio (ie. amounts of insert and amounts of vector recommended by your website). Presently, I've just been overloading my rxn mix volume to the max with megaprimer and no water because I was told you need a very high insert to vector ratio. Only question is how did you get these insert: vector ratio recommended on your website?

Any advice would be much appreciated!