Field of Science

Transformation by DNA on beads

Yesterday I incubated some of my competent H. influenzae cells with DNA carrying a novobiocin-resistance gene.  One tube got the normal prep of chromosomal NovR DNA.  Two others got DNA that had been cut with one of two restriction enzymes (either EcoRI, average fragment size 6 kb or XhoI, average fragment size 12 kb) and then had biotin incorporated at the ends of the fragments, and three others got streptavidin-coated polystyrene beads with the biotinylated DNA bound to them.

I had calculated that the first batch of DNA+beads had about 250 ng of DNA per ml, so I used the same volumes of beads for the others, and diluted the non-bead DNAs to 250 ng/ml before using the same volumes.  I incubated the DNA plus cells for 15 minutes and then plated the cells on Nov agar, and (diluted) on plain agar.  Today I counted the colonies and calculated the transformation frequencies.

Point 1.  Cutting the DNA with XhoI reduces its transforming ability by 4-fold, but cutting with EcoRI reduces it by about 100-fold.  So I checked where these two enzymes cut relative to the NovR (gyrB) gene.  EcoRI cuts inside the gene, but XhoI only cuts at sites 1 kb and 3 kb on either side of it. 

Point 2.  DNA attached to beads transforms!  This wasn't a sure thing, for lots of reasons.


Point 3.  DNA attached to beads transforms 10-30-fold worse than the same amount of free DNA.  This is not surprising because most of the DNA on the beads will be inaccessible because it's tangled up with or behind other DNA fragments.

Now I'll celebrate by spreading some poly-L-lysine on some cover slips, using the new method I was taught yesterday, so tomorrow I can test whether cells bind to these and whether they stay alive after binding.

1 comment:

  1. Clem WeidenbennerMay 22, 2010 at 8:59 AM

    Rosie:
    If EcoR1 cuts inside the gene, then where do the Nov resistant transformants come from? Are they taking up multiple pieces of DNA and making a functional gene through repair? Is the EcoR1 digestion incomplete?

    ReplyDelete

Markup Key:
- <b>bold</b> = bold
- <i>italic</i> = italic
- <a href="http://www.fieldofscience.com/">FoS</a> = FoS