Dot blot on bacteria

Mad Mat hauwelm at minitel.net
Thu Apr 8 15:53:12 EST 2004


I sometimes screen bacterial clones by Southern-blot after cloning of
DNA fragments. The lysis protocol could be suitable for your
application.
I grow bugs in Terrific Broth agar so the colonies are fat and juicy.
Each clone is in duplicate (I grow up to 70 clones per Petri dish). I
then apply a nitrocellulose membrane to the dish and proceed to the
lysis (dont forget to draw marks on the membrane and on the dish, I
know it sounds obvious but...)

Fill three large weighing boats with respectively denaturing buffer
(1.5 M NaCl, 0.5 M NaOH), neutralising buffer (0.5 M Tris-Cl, 1.5 M
NaCl, pH 7.4) and  washing buffer (10X SSC: 1.5 M NaCl, 0.15 M Tri
sodium citrate)
Place carefully the membrane on the surface of the lysing bath using
plastic forceps with colonies facing up. Leave a couple of minutes,
shake the bath gently to sink the membrane and leave another five
minutes. Use plastic forceps to take the membrane out and make it
glide against the side of the boat to eliminate the excess of buffer
Repeat this operation for the other two baths
Let the membrane dry on Whatman paper (colonies facing up)
Fix the proteins to the membrane by UV cross-linking 

The colonies might come off during the baths, it is not a problem as
long as you avoid cross contamination of the spots.

hope this will work for you

good luck

MM



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