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forgot to heat shock


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#1 lyok

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Posted 18 March 2014 - 01:36 AM

Dear all,

 

I forgot to do a heat shock when transforming e.coli. I just had the cells on ice (after adding competent cells to the plasmids) for 15 minutes and then I had to do the heat shock followed by adding the LB medium. However I forgot to do the heatshock.

 

Now I wonder: has anyone done this before? Do you still have growth?

I am going to simple use the cells anyway, to see if it will still work.. (and I cant redo it anyway at the moment), but just curious if anyone had a similar problem.

Theoretically one might say it could still work.. but curious you ever had a similar problem.

 

 



#2 hobglobin

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Posted 18 March 2014 - 10:55 AM

If I remember right we had once a problem with a water bath apparatus which couldn't keep a constant temperature...the transformation efficacy was just very low and almost no colonies finally.

 

And moved it to cloning.


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#3 Trof

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Posted 19 March 2014 - 07:53 AM

I'd like to hear about the result, but my guess is.. uhm, nope.


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#4 lyok

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Posted 20 March 2014 - 03:57 AM

I'd like to hear about the result, but my guess is.. uhm, nope.

 

Well.... all samples "worked". So I could use them. The number of transformed cells were lower (a lot), but I still had enough cells to continue!



#5 Trof

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Posted 20 March 2014 - 07:42 AM

Interesting.

Plasmid size? Ligated (how?) or just re-transformation?

What kind of competent cells?


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#6 lyok

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Posted 20 March 2014 - 10:45 AM

Interesting.

Plasmid size? Ligated (how?) or just re-transformation?

What kind of competent cells?

10-12kb.

ligated? It was after an LR reaction! (gateway reaction).


So the plasmid was not cut or ligated.

 

And it were the typical top10 chemical competent cells.



#7 Trof

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Posted 20 March 2014 - 02:19 PM

ligated? It was after an LR reaction! (gateway reaction).


So the plasmid was not cut or ligated.

 

And it were the typical top10 chemical competent cells.

 

 

 Technically the plasmid was cut and re-attached by clonase enzyme mix, but important is that the plasmid was probably intact at time of transformation.

But this completes the information, thanks. 


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon





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