hi
I am trying to electroporate B. subtilis (168 got from BGSC) cells. I
am yet to get any transformants. I need to use the electroporation
technique and have already tried Brigidi's et al procedure (using PEB
buffer, containing 272mM sucrose, 1mM Mgcl2, 7mM pot. po4. ph 7.4) after
washing the pellet with 1mM HEPES. I used 0.5ug of DNA per 400ul of cells
without pulse controller at 6250V/cm, 25uF. I have tried doubling the
voltage to 2.5kv /0.2 cm cuvette. I have tried using both 0.1cm and 0.2 cm
cuvettes. I don't have any 0.4 cm and above cuvettes. I have also tried
Matsuno's et al., procedure which is quite similar except that they use a EB
buffer containing PEG. But I yet to see any transformants. I have tried
increasing and decreasing DNA concentration. All work has been done ona
Biorad's gene pulser II equipment fitted with a pulse controller and a
extented capacitance unit. I am using two types of plasmids, pBC16 (4.6kb)
and another 9.1kb plasmid. Both --no results. By the way, I have also the
natural competency technique a few times, with no results even though I
haven't yet used pBC16.
I have been trying for a few months now. Please somebody help me since I
am at my wits end.
thanking any good samaritans who come forward.
cheers
jakku
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||R. JAYAKUMAR
CSIR- Senior Research Fellow
Dept. of Molecular Microbiology,
School of Biotechnology,
Madurai Kamaraj University,
Madurai - 625 021.
India
email: jakku at linuxfan.com
tel: +91-452-858471-374
efax: (603)-688-4665
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||"YOu harvest what you sow.. so sow happiness everywhere"
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