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Fake positive colony in lamda RED reconbination, why?


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#1 MXH

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Posted 02 April 2010 - 04:53 PM

Hi all,

Recently, I perform a number of pflB-focA gene knock-out using lamda RED recombination in Escherichia coli strain W1485.
But I have a hard problem.
Below is my experiment:
1. PCR amplify linear fragment from both pKD3 and pKD4
Purify PCR product by gel.
2. Make target W1485strain maintaining pKD46 by growing at 30C with 10 mM L-arabinose (E. coli was induced for just 1 h before harvesting)
3. Transform E. coli using Calcium Chlorid.
4. Plate the transformation culture on LB plates supplemented with chloramphenicol (8ug/ml) or kanamycin(40ug/ml).

Here is the results:
1. I can get several dozen colonies from plate with kanamycin, but cann't get any colony from chloramphenicol. :P :P :(
2. I cann't amplify anti-kanamycin gene from these anti-kanamycin strain. :) :blink: ;)
Can anyone tell me what could be the problem.


Regards

Ma

Edited by MXH, 02 April 2010 - 04:55 PM.


#2 MXH

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Posted 02 April 2010 - 05:09 PM

During RED recombination, is electroporation necessary?


Hi all,

Recently, I perform a number of pflB-focA gene knock-out using lamda RED recombination in Escherichia coli strain W1485.
But I have a hard problem.
Below is my experiment:
1. PCR amplify linear fragment from both pKD3 and pKD4
Purify PCR product by gel.
2. Make target W1485strain maintaining pKD46 by growing at 30C with 10 mM L-arabinose (E. coli was induced for just 1 h before harvesting)
3. Transform E. coli using Calcium Chlorid.
4. Plate the transformation culture on LB plates supplemented with chloramphenicol (8ug/ml) or kanamycin(40ug/ml).

Here is the results:
1. I can get several dozen colonies from plate with kanamycin, but cann't get any colony from chloramphenicol. :P :P :(
2. I cann't amplify anti-kanamycin gene from these anti-kanamycin strain. :) :blink: ;)
Can anyone tell me what could be the problem.


Regards

Ma



#3 Micro

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Posted 05 April 2010 - 08:20 PM

Hi MXH,
I don't know the answer, but this question might get a few more responses if you re-posted it in the "Molecular Cloning" heading rather than "Microbiology".

Cheers
M :(

#4 MXH

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Posted 06 April 2010 - 04:03 PM

Hi MXH,
I don't know the answer, but this question might get a few more responses if you re-posted it in the "Molecular Cloning" heading rather than "Microbiology".

Cheers
M :lol:



Thank you for your suggestion, Dr. M
I'll try it




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