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.
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 4o and 16o 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!