Do we need to do gel extraction before proceeding to ligation step with insert?
My understanding is that antarctic phosphatase treatment is mandatory while we are digesting plasmid with single enzyme. Correct me if I am wrong
No need unless you are using a pair of enzymes that generate compatible ends, for example as in the case of NheI and SpeI.
You can skip gel extraction per se and just purify the insert with gel extraction kit.
Hi there!
I feel treating with Antarctic phosphatase will increase your efficiency of cloning, because it reduces the chances of vector-vector ligation manifold. It happens at times that vectors get ligated to each other without any insert and also enters the competent cells (even though they are double the usual size and transformation efficiency should be less) when you use the ligated mix for transformation. So it is better to remove phosphate and then use phenol-chloroform-isoamyl alcohol to remove the enzyme and precipitate the DNA with ethanol. You can then use it for ligation.
Hope it helps!
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