Cloning Catastrophe

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Ok, scientific community, I come to you in desperation far later than I should have. I can’t clone a cohort of fusion protein DNAs that I absolutely must. The screwed up part: it’s basic cloning. I’m no novice to this, but I feel like one at this point. Here’s what I’ve got:

Insert and vector both double digested with XbaI and SpeI (yes, I know they’re compatible).
I’ve treated the cut vector with alkaline phosphatase and column-purified it.
I’ve tried 2 different AP treatments, 2 different ligases, and 2 different types of competent cells, and fresh plates every time.

All of this leads to one reproducible result: blank plates. Positive control plates flourish, but I might as well be plating Clorox on the experimental plates. Can anyone suggest something? My job may depend on it :)


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12 thoughts on “Cloning Catastrophe”

  1. ive been having similar troubles with my subcloning and after a couple of months one of the protocols i was trying worked

    1) shortening AP treatment
    2) digesting with more enzyme
    3) ligating with more DNA at incredibly high ratios

    this helped with a couple of my constructs in pProEX but still not working for my pFastbac .. so i might try my phosphatase treatment for an even shorter time

    there are some good ideas here and im glad i ran into this blog entry

    also, i use TOP10 competent cells from invitrogen, which i believe are super sensitive to mechanical lysis, so i think ill try shaking at slower speeds even though i was doing it at the standard 225-250 rpm

    expression of my insert is toxic for the bacterial cells but i had never thought of it as potentially killing my competent cells. i dont know if that is the problem coz i use SOC medium which has some glucose in it…

  2. Hi all..I am new to this blug.. have gone through it lots of time its great one you guys are doin gr8 job.

    I am ligating BamHI cut vector pLAFR3 (SAP phosphorylated) with SAU3AI cut DNA (~20kb) and transformation in HB101, but getting no clonies. I have tried different vector insert ratio but nothing is working.

    Wts wrong goin on.. got no idea..

    All of your help and comment would be highly appriciated.

    Thanks

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