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White colonies no insert


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#1 newbie2010

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Posted 24 November 2010 - 02:38 PM

Hi
I'm new in molecular biology. I did a transformation and I've got white colonies which I've screened. However there is no insert in the white colonies I've picked. They are pure white (no blue in centre/light blue). I've kept the plates in 4C for 3h after 37C for 16h/overnight. Pls help.

#2 Chakchel

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Posted 24 November 2010 - 11:25 PM

Hi,
did you check the plasmid size on a gel?
When you compare them to the empty vector (blue colony as control) you might see that your negative white colonies are a little bit smaller than the blue one.
Sometimes breaks with loss of a piece of the lac-a region occurs, leading to white colonies because the lac-a is not active anymore as it also would be when you insert your piece of interest into this region.

#3 HomeBrew

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Posted 25 November 2010 - 03:36 AM

We will need more information to help you find where your cloning experiment might have failed. How did you prepare your vector and insert? What enzymes did you use? Did you gel purify your fragments? How did you do your ligation? How did you prepare your competent cells? What controls did you use? What antibiotic are you selecting with? Did you add xgal (or some other chromogenic substance) to the plates?

There are many points at which a cloning experiment can fail. Given only that you "got white colonies with no insert", all we can say is that your cloning experiment failed...




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