We consistently get good results without heat-treating the bacteria at
all.

1.  Grow Ka or Raoultella planticola (nonbiohazard) on SM plates, 48-72
hours at room temperature.

2.  Pretreat the transfected Dicty cells overnight with 10ug/ml
blasticidin in HL5.

3.  Resuspend the bacteria in KK2, using 2.5ml buffer for each SM plate.

4.  Spread 0.5 ml of the bacteria suspension, together with the pretreated
Dicty cells, on KK2 plates containing 40ug/ml blasticidin.  20ml B40 agar
per plate is plenty.

Blasticidin-resistant Dicties form clones after 3-4 days with high
efficiency.  The plates can be loaded with at least 3e6 nonresistant
amebae without any growthrough, provided the cells have been pretreated
with blasticidin.

best wishes,

Harry MacWilliams
--------------------------



On Tue, 9 Mar 2010, prasad ls wrote:

> Dear Dicty colleagues,
>
> We are doing REMI transformation of Dicty and select on plates with autoclaved bacteria (K. aerogens). We are unable to spot the colonies due to clumping of dead bacteria. This is not due to drying either as the plates are kept reasonably moist. Is there a way to overcome this problem of clumping? Any suggestions will be great to have. Thanks in advance.
>
>
>
> Prasad
>
> IIT Madras
> Chennai,INDIA.

----------------------------

Prof. Harry K. MacWilliams
Biologiezentrum der Ludwig-Maximilians-Universität
Grosshadenerstrasse 2
82152 Planegg-Martinsried
GERMANY

tel (office) +89 2180 74 288
tel (lab)    +89 2180 74 289
fax.         +89 2180 74 219

callers from North America please dial:
tel (office) 0114989 2180 74 288
tel (lab)    0114989 2180 74 289
fax          0114989 2180 74 219