if after cutting i realize that the digestion (and heat inactivation if enzymes) isn't complete, can i again add the same enzyme and continue the digestion reaction?
Hi there,
Sure you can! The heat inactivation inactivates the restriction enzyme by denaturation. The denatured protein is no longer able to bind to its specific restriction site as it has lost its native form. So there will be no competition for the restriction site between the inactivated enzyme and the native enzyme. But if you want to work in a more rigourous way, you should first purify the whole DNA from the sample (using silica column) and then perform a new digest on the eluted DNA.
Hello Ayda,
To answer Yuhong's comment, I suppose that you analyzed an aliquot of your reaction mixture by gel electrophoresis and that's how you found that digestion wan't complete. Yes, you can add another batch of enzyme and proceed with further digestion of your sample, but take care that the final concentration of glycerol (coming from the restriction enzyme stock) doesn't exceed 10 % in order to avoid problems due to star activity. If necessary, you can add more digestion buffer to the digestion mixture to fulfil this requirement.
Ayda Assadi , It can be done, but if you want to describe the results of your research with equations, it is better to repeat the experiment for a longer time. Unfortunately, this is an additional cost.
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