Electroporation Protocol for Nostoc punctiforme                                                                    5/2000

Preparation of Nostoc:

         a.  Concentrate the Nostoc culture by centrifugation and resuspension in 10-20 ml of medium.  Sonicate
         the suspension briefly (we use 4 x 10 sec bursts at 50% duty with a power output of 3 using the microtip 
         of a Heat Systems Sonicator; sterilize with ethanol); this step seems to be essential.  Pellet and resuspend 
         the cells in medium with nitrogen supplementation and allow them to recover for 4 h to overnight.

         b.  Prior to electroporation, wash the cells 4 x with 20 ml room temperature sterile ddw for each wash.  
         We have found that cold water will reduce viability of cells following electroporation.  Resuspend the cells 
         in a final volume at a concentration of 50-100ug Chl a/ml in ddw (1ug Chl a ~ 106 cells).  

Preparation of DNA:

         Approximately 10 ug DNA/electroporation is recommended.  Less DNA can be used with decreased
         efficiency.  We typically Qiagen prep the DNA and resuspend the DNA in ddw.  DNA that is not as clean
         or has contaminating salts will also decrease the efficiency or cause arcing during electroporation.

Electroporation:

         a.  Aliquot DNA to sterile microfuge tubes and set on ice.  Chill the elctroporation cuvettes on ice. 
         Add 400 ul of concentrated washed cells to the microfuge tube.  Mix by pipetting gently up and down.  
         Transfer to cold 0.2 cm electroporation cuvettes, cover and electroporate with the following parameters:
         600 ohms, 1.6 kEV, 25 mF.  Expected time constants should be about 11-13.

         b.  Immediately following electroporation, dilute the cells with 600 ul of medium supplemented with 
         2.5mM NH4Cl + 5 mM MOPS + 20 mM MgCl2 (or equimolar MgSO4 and MgCl2).  Transfer the cells 
         into a recovery flask containing the same medium.  Incubate under dim light with gentle shaking overnight
         to allow for recovery.

         c.  The cells can now be concentrated and plated onto selective media or placed under liquid selection. 
         We normally split each sample and plate some of the cells and leave the remaining in liquid.  Resistant
         colonies on plates should be visible under a dissecting microscope within 14 days.

         d.  Antibiotic concentrations used in Nostoc:
         for pSCR19 based plasmids:  10 ug neomycin/ml
         for pSCR202 based plasmids:  10 ug ampicillin/ml