Fake positive colony in lamda RED reconbination, why?
April 3, 2010 at 1:14 am #13046
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?
April 5, 2010 at 5:56 pm #98804
can you put the colonies to another plate (or liquid media), so that they were growing?
April 7, 2010 at 12:10 am #98835
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.
April 7, 2010 at 5:55 am #98841
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?
April 8, 2010 at 3:16 am #98859
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.
April 8, 2010 at 7:51 am #98862
what do you mean by "like anti-kanamycin gene"? Did you amplify the same gene?
April 8, 2010 at 8:06 am #98863
By use these words, "like anti-Kanamycin gene", I mean the size of isocitrate lyase gene is similar to anti-Kanamycin gene
April 8, 2010 at 8:21 am #98864
the size doesn’t matter. The point is, whether your primers work with your PCR conditions
April 8, 2010 at 9:04 am #98865quote JackBean:
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.
April 8, 2010 at 9:31 am #98867
your primers can anneal two genes?
April 8, 2010 at 10:32 am #98868quote JackBean:
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.
April 8, 2010 at 10:41 am #98870
I see. Do you have some positive control for experiment 2 with primers B?
April 8, 2010 at 11:13 am #98871quote JackBean:
Actually, I want knockout eight genes.
And I transform 16 PCR products (eight of them are amplified using PKD3 as templete, and others are amplified using PKD4) to 16 competent cell. I did this at least 5 times. None of them seems work. 🙁
I did every thing I can do, such as using different L-ara concentration to induce RED recombination enzymes, using different PCR product concentration to transform competent cell, using different drug (kanamycin or chloramphenicol) concentration.
The results are still "I can get several dozen colonies from plate with kanamycin, but cann’t get any colony from chloramphenicol.
" and "I cann’t amplify anti-kanamycin gene from these anti-kanamycin strain."
I need suggestionssss……
April 8, 2010 at 12:00 pm #98872
OK, take your plasmid, which you use for transformation and try the PCR to see, whether your primers B work with your conditions 😉
BTW in biology we use false positive, not fake 😉
April 9, 2010 at 1:01 am #98875quote JackBean:
Your "BTW" is very constructive. Thank you, JackBean
You are the "JackBean" to me 😆
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