Fake positive colony in lamda RED reconbination, why?

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    • #13046
      MXH
      Participant

      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

    • #98804
      JackBean
      Participant

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

    • #98835
      MXH
      Participant

      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.

    • #98841
      JackBean
      Participant

      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?

    • #98859
      MXH
      Participant

      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.

    • #98862
      JackBean
      Participant

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

    • #98863
      MXH
      Participant

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

    • #98864
      JackBean
      Participant

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

    • #98865
      MXH
      Participant
      quote JackBean:

      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.

    • #98867
      JackBean
      Participant

      your primers can anneal two genes?

    • #98868
      MXH
      Participant
      quote JackBean:

      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.

    • #98870
      JackBean
      Participant

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

    • #98871
      MXH
      Participant
      quote JackBean:

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

      No
      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……

    • #98872
      JackBean
      Participant

      OK, take your plasmid, which you use for transformation and try the PCR to see, whether your primers B work with your conditions 😉

      EDIT
      BTW in biology we use false positive, not fake 😉

    • #98875
      MXH
      Participant
      quote JackBean:

      OK, take your plasmid, which you use for transformation and try the PCR to see, whether your primers B work with your conditions 😉

      EDIT
      BTW in biology we use false positive, not fake 😉

      Your "BTW" is very constructive. Thank you, JackBean
      You are the "JackBean" to me 😆

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