Shooting Trouble During Cloning

As frustration goes, cloning is often up there with trying to thread a camel through the eye of a needle.

You do everything carefully: prepare your vector and fragment DNA, cut them as the restriction enzyme manufacturer instructs (complete digest in five minutes!), ligate, transform and go home in anticipation of a good number of clones on the cloning plate and zero on controls.

You already have the plan of using the resulting construct to do The Real Experiment – a protein purification or localization study. But the next morning you see a disaster: no clones or too many clones and you stand there trying to understand which of the multiple steps went wrong.

No fear, we will help you with a little diagram.

Let me explain.

Start with vector + fragment and two controls – uncut vector and cut vector.

• Uncut vector is a control of your competent cells and transformation efficiency. No clones with this means something is wrong with either or both, but your cloning could be fine.
• Cut vector is a control of your digest and, in case of compatible ends, vector dephosphorylation. Clones with this control suggests you have incomplete digestion or dephosphorylation.
• If “vector + fragment “ produces no clones, there a couple of things you can check. Check your transformation using the uncut vector control. If transformation is fine, check cut vector control. Clones with this control could mean you had an incomplete digest. To fix this, change digest time and/or DNA concentration. (Five-minute digests and twenty-minute ligations never work in my hands.)
• If “vector + fragment” produces only empty vector clones, most likely something is wrong with your fragment. Check the quality and concentration of your fragment DNA and make sure you have complete digestion of fragment.

Of course, this is not an exhaustive scheme. For example, your transformation may not work because of a dummy E.coli aliquot, or because while your water bath display says it is 42°C it is really only 30°C. Or your vector is not ampicillin but kanamycin resistant and you forgot.

What I hope you get from my diagram is that when things go wrong, carefully deconstruct and put together each stage of cloning to find where the issue lies.

Happy cloning.

Vicki Doronina

Vicki has a PhD in Molecular biology from the University of Edinburgh.

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