Thanks for the replies.

Best wishes,

William S. Swatson
Graduate Student
Department of Biological Sciences
University of Missouri, Columbia
________________________________________
From: DICTY [[log in to unmask]] on behalf of Michael Myre [[log in to unmask]]
Sent: Friday, December 20, 2013 3:56 PM
To: [log in to unmask]
Subject: Re: [DICTY] Dictyostelium Transformation

Biorad genepulser:
0.8-0.9 kV...usually 0.85
25 uF
200 ohms
0.1cm cuvette

100 ul of cells suspended in a slurry of DNA that was eluted in
electroporation buffer.

two pulses....5 sec between pulses...0.6-0.9 time constant.

healing solution is optional but i use it...a mix of CaCl2 and
MgCl2...I add 2 uL to the mix post-transformation. 10 minutes on ice.
dilute in fresh HL5 - use antibiotic as a prophylactic...not AMP ever.
especially if AMP resistance resides on the vector you used. use Kan.
plate diluted mixes into 4-5 10 cm dishes and incubate at 21-22C
overnight.
add drug the next day (e.g., G418) after 16-20hrs recovery. do a kill
curve first if the drug is pre-made and fresh. GFP-expressing cells can
be seen at this time prior to adding the G418.

with this technique...a single confluent well from a 6-well dish and
1-2 ug linearized DNA eluted in electroporation buffer will give you
thousands of colonies

On 2013-12-20 16:01, Swatson, William S. (MU-Student) wrote:
> Dear all,
>
> I have been trying to perform targeted gene disruption by homologous
> recombination in AX4 cells using electroporation with no success. The
> conditions for electroporation differ in almost every protocol I have
> laid hands on. This is probably owing to the different apparatus
> being
> used. We use a BIO-RAD gene pulser with the following conditions; 1
> kV, 3 uF and 200 ohms. We use 0.4 cm cuvettes. Specific questions
> are,
> what conditions have been used successfully using this type of
> apparatus? Is a resistor necessary? Is it better to perform a single
> pulse or an additional pulse? I welcome any suggestions. Thank you
> and
> happy holidays.
>
> William S. Swatson
> Graduate Student
> Department of Biological Sciences
> University of Missouri, Columbia

--
-------------------------------------------------
Michael A. Myre, PhD
Instructor of Neurology, Asst. in Genetics
Center for Human Genetic Research
Massachusetts General Hospital
Harvard Medical School
185 Cambridge St. Rm. 5.612A
Boston, MA 02114
Ph. 617-643-5536
[log in to unmask]
Website: http://chgr.org/index-faculty_myre.html
Website:
http://www.massgeneral.org/neurology/researcher_profiles/myre_michael.aspx
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