help with transformation! thanks!! - (Mar/26/2009 )

100% with Homebrew. Switch immediately to normal ligase and (fresh) ligase buffer. Make sure your gel isolation isn't toasting your DNA with short wave UV. Measure your competency by transforming 1 ul of a 10 pg/ul dilution of a known working plasmid.

Thanks, I will try the regular ligation. My cells are competent; I've tested them with my plasmid.

Transforming with plasmid miniprep DNA will almost always work. You need higher competence than can be tested that way -- you need to check with high dilutions of plasmid DNA.

I tried overnight ligations at 4^o and 16^o with regular ligation. There were more colonies on the plates than with the Quick Ligation, but the negative control still has the most colonies. Maybe I can use phosphatase treatment with the regular ligation? I am testing the competency of my cells today. If I get a lot of colonies doing a regular ligation, does this indicate that my cells are competent, or do I still need to use the pUC19?

Are you checking any of the clones that come up on the non-control plates? Is this a situation where you just need one correct clone, or do you need many? I can't recall which, but one of the "X" enzymes (Xba or Xho) always gave me trouble. Are there alternative enzyme(s) you can use to clone?

I just need one, although in the past when I've gotten more colonies on my negative control, none of the other colonies turn out to be right. I also tested my cell competency by transforming 1ul of 10pg/ul pUC19 into TOP10 cells, following the provided protocol from Invitrogen. I only got about 10-15 colonies though, instead of the 100 that is predicted. Do I need to alter my transformation protocol to improve efficiency? Thanks!