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Making Competent Cell

About microscopic forms of life, including Bacteria, Archea, protozoans, algae and fungi. Topics relating to viruses, viroids and prions also belong here.

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Making Competent Cell

Postby kenpier » Tue Sep 06, 2011 10:11 am

Hi,

Anyone here have a good protocol in making chemical treated competent cells fast?

Currently using rubidium chloride method.
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Postby canalon » Wed Sep 07, 2011 11:53 am

RbCl is exactly what I use when I want to obtain chemically competent cells quickly with a good efficiency.
Following a protocol similar to this one: http://130.15.90.245/e__coli_competent_cells.htm

What is wrong with it?
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Postby kenpier » Thu Sep 08, 2011 3:18 am

The competency level of the cell don't always hit 1x10exp8 to 1x10exp9 cfu's/ug.

Sometimes is just too low, result are not always consistent. Is there anything to take note?
I know that TFb1 and TFb2 need to dissolve individually before mixing.
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Postby canalon » Thu Sep 08, 2011 5:07 pm

I must say that I never measured the competency level, and in most case got my transformants (as for most of the other cases, that is common when you try to express a protein that is toxic for your bacteria).
I have been known to "steal" from TA cloning kits the competent cells that came in the package (we had a lot of that because generally they were not what we wanted to transform our constructs in) as they are a bit more consistently successful.

But for TFb1 and 2, they were prepared in a batch in advance and stored in the fridge fully dissolved and ready to use (they are really stable).
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Postby kenpier » Tue Sep 27, 2011 5:33 am

When preparation of competent cell, which freezing well be better?
Dry Ice or Liquid N2?
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Postby JackBean » Tue Sep 27, 2011 5:53 am

the cells need to be frozen as fast as possible, so that the water crystals will be small. For that reason is used nitrogen usually.
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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