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"competentcy" of my method


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#1 wizzkid

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Posted 12 March 2010 - 05:04 AM

hi,

I transformed 3 identical plasmids into same strain of competent cells and incubated overnight at 37 degrees. I came back the following morning and found that only one of my plates contained colonies.
Could the failure of the other plates be to the competent cells i happened to transform them in? or prehaps during the tranformation process after spinning the bacteria cells down (after 20 mins in shaking incubator) i failed to resuspend the pellet in SOC medium.. could someone prehaps add an opinion?

Danke!

#2 vladooo

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Posted 23 March 2010 - 08:49 AM

Anything could happen. But I observed that thawed competent cells are settled down and when you pippete them from the top there can be minimum of cells in the first aliquots. To avoid this, pippete them up and down gently before aliqoting.
If you are using single-packaged competent cells ignore this post entirely.

#3 lucilius

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Posted 23 March 2010 - 09:53 AM

just a quick question: single packaged competent cells, are those competent cells packed 1 by 1 ? How do they do that and whats the point of working with 1 cell?

#4 vladooo

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Posted 23 March 2010 - 10:52 AM

It's not one cell :D It is one aliquote (for example 20 ul) of competent cell. You add just your ligation in every single tube.
BTW I never buy competent cells, it's sooo expensive - I do my own DH5a this way (http://cse.fra.affrc...tentCell-e.html) and it works great. I store them at -80C and they are good for at least 4 months.

#5 lucilius

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Posted 02 April 2010 - 05:35 AM

It's not one cell It is one aliquote (for example 20 ul) of competent cell. You add just your ligation in every single tube.
BTW I never buy competent cells, it's sooo expensive - I do my own DH5a this way (http://cse.fra.affrc...tentCell-e.html) and it works great. I store them at -80C and they are good for at least 4 months.


Ah I see, thanks.

Anything could happen. But I observed that thawed competent cells are settled down and when you pippete them from the top there can be minimum of cells in the first aliquots. To avoid this, pippete them up and down gently before aliqoting.
If you are using single-packaged competent cells ignore this post entirely.


One more question: why isnt this a problem in single packaged cells? Arent they also at the bottom? When you buy them, they have been stored for long too , so havent then settled down too?

#6 snoopyx

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Posted 02 April 2010 - 03:59 PM

Yes they have. But it's about the total number in one aliqout. It'll be the same. If you aliqout them on your own it can happen (tom em as well) that the first aliqouts contain less cells ergo less cell that can be transformed ergo less colonies on a plate.




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