Experimental Protocols

Meeks Laboratory

 
Protocol for PCR from single colonies                                                                             5/2000

           This protocol was developed for isolating DNA from small amounts of Nostoc punctiforme biomass for
PCR purposes.  The cells can be colonies from a plate or a small amount of cells growing in liquid.  A small 
amount of agar contamination does not seem to be a problem.

1.  Pick colonies from plate and place in a microfuge tube, or pipette a small amount of cells into a microfuge tube.
Pellet cells and resuspend with 200 ul of TE, pH = 8, + 1% Triton X-100.  Pipette up and down vigorously to mix.

2.  Heat tubes at 95C for 3.5 min.  The mixture may become cloudy.  This is OK.

3.  Extract twice with 200 ul of chloroform each time.  Centrifuge for 2 min at maximum in a microfuge to separate
the layers.

4.  Use 10 ul of the aqueous phase in 100  ul of  a standard PCR reaction
 
ddw
 
49.5 ul
 
DNA from aqueous phase
 
10
 
10X PCR buffer
 
10
 
5 pmol/ml primer 1
 
10
 
5 pmol/ml primer 2
 
10
 
10 mMdNTP
 
2
 
25 mM MgCl2
 
8
 
AmpliTaq polymerase
 
0.5
 
PCR cycles:
           1  cycle: 5 min 94C, 1 min 50C, 4 min 70C
           30 cycles:  1 min 94C, 1 min 50C, 4 min 70C

Reference:  Hagen and Meeks. 1999. J. Bacteriology 181:4430-4434.