Transformation troubles

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    • #9175
      mmustafa
      Participant

      Hi All!!

      I am struggling with trying to get my plasmid into competent cells. I am currently using the Invitrogen Gateway Directional TOPO kits and One Shot TOP 10 competent cells and have had no success with the transformation process. Any clues anyone?????

    • #82285
      Cat
      Participant

      How about +ve control???

    • #82287
      mmustafa
      Participant

      I used the control plasmid provided with the competent cells. No colonies there either!!

    • #82289
      Cat
      Participant

      O.K., you have too many things that can go wrong – anything in the procedure to bad cells. The most common problems: 1. Heat shock: too long, wrong temp; 2. wrong antibiotic in selection media. If you are sure that you did not make mistakes in the procedure, then you can assume bad cells.

    • #82293
      mmustafa
      Participant

      I have been heat shocking for 30 to 45 sec at 42C. That should be okay, right?

    • #82294
      Cat
      Participant

      That should be fine. Check the procedure step by step and see if you are doing anything different:

      http://www.invitrogen.com/content/sfs/m … 10_man.pdf

    • #82296
      mmustafa
      Participant

      Nope! Nothing’s different!!!!

    • #82297
      Cat
      Participant

      Then your problem is with competent cells.

    • #82308
      mmustafa
      Participant

      I got new stock of competent cells and performed transformation using a pUC19 control plasmid and got really tiny colonies. What does that mean??

    • #82311
      Cat
      Participant

      If there are many small colonies, then your transformation probably worked and size problem is likely due to too much antibiotic in the media. If there are only few small colonies, then they are likely to be escapees…

    • #82325
      h2so4hurts
      Participant

      Haha, or he didn’t let the plates grow long enough…

      Also, try adding 4uL of your PCR product into to TOPO reaction, especially if your PCR product is weak. Maybe try gel purifying your PCR product before the TOPO reaction.

    • #82344
      mmustafa
      Participant

      I got really nice strong single band (correct size) when I ran my PCR product on a gel. That’s why I was confident that the transformation would go smoothly!!

    • #82483
      xixie
      Participant

      hehe , i agree with h2so4hurts,who says the colony do not grow long enough ,in my opinion , top 10 cell should grow more than 16h,but DH5a just need overnight (12h-14h).

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