Fake positive colony in lamda RED reconbination, why?

[MXH]

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 30°C 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.
2. I cann't amplify anti-kanamycin gene from these anti-kanamycin strain.
Can anyone tell me what could be the problem.
During RED recombination, is electroporation necessary?


Regards

Ma

[JackBean]

can you put the colonies to another plate (or liquid media), so that they were growing?

[MXH]

Thank you Dr. Jackbean

I have put these colonies to both kanamycin plate and kanamycine LB liquid medium.
They can growth on these medium. I purified their genome DNA, and cann't amplify anti-kanamycin gene
This confuse me.

[JackBean]

Can you amplify the gene from some positive control (like your plasmid before insertion) with the same conditions?
Did you try several dilutions of the gDNA?

[MXH]

Thank you, JackBean

I can amplify the isocitrate lyase gene (1.3kb, like anti-Kanamycin gene)from all positive control with the same conditions.
But I cann't amplify anti-Kanamycin gene from all positive colonies.

I was frustrated.

[JackBean]

what do you mean by "like anti-kanamycin gene"? Did you amplify the same gene?

[MXH]

Sorry,
By use these words, "like anti-Kanamycin gene", I mean the size of isocitrate lyase gene is similar to anti-Kanamycin gene

[JackBean]

the size doesn't matter. The point is, whether your primers work with your PCR conditions

[MXH]

the size doesn't matter. The point is, whether your primers work with your PCR conditions

Yes, I say this because I do a control experiment:

Same primers, same PCR conditions. The only exception is that the templete of control experiment is PKD46.

[JackBean]

your primers can anneal two genes?

[MXH]

your primers can anneal two genes?

Sorry, I didn't exactly express my opinion.
I do two control experiments:
1, Amplify isocitrate lyase (using primer pairs A), to test whether the genome templete is OK.
2, Amplify anti-kanamycin gene using PKD46 as templete (using primer pairs B), to test whether the PCR system is OK.

[JackBean]

I see. Do you have some positive control for experiment 2 with primers B?