I need to digest my plasmid using BamHI and SacI. Unfortunately, the enzymes came from different manufacturer, Fermentas and Wako Nippongene respectively. I checked the buffer ingredients and they are quite different. I want to do double digestion by first digesting my plasmid using SacI then BamHI since the latter needs higher salt concentration. Any thoughts regarding this matter?
I would not recommend mixing two buffers from two different manufacturers. Their ingredients can vary significantly and you are not likely to get a good digest out of that. Instead, I would recommend a single digestion with one of the restriction enzymes followed by DNA purification; then you can perform the second digestion with the purified DNA.
It is better to use the same manufacture because of its ingredients. You can use the different manufacture but the efficiency of digestion is not 100% so you obtain partial digestion.
best regards
No you cannot mix two buffers from different manufacturers for performing double digestion. Even you cannot mix two different buffers from same manufacturers for double digestion with the enzymes whose buffer requirements are totally different. It is better to use single buffer even if the efficiency of digestion is less than 100% for both restriction enzymes.
I would not recommend mixing two buffers from two different manufacturers. Their ingredients can vary significantly and you are not likely to get a good digest out of that. Instead, I would recommend a single digestion with one of the restriction enzymes followed by DNA purification; then you can perform the second digestion with the purified DNA.
If you go to the manufacturer's website it will tell you what other buffers you can use (and just pick the highest percentage for your different enzymes) but it's not recommended to use buffers from different manufacturers.
Hi
I agree with Juan... You can try these enzymes' compatibility in various digestion buffers and select the most suitable buffer. However... If you are preparing vector backbone for cloning then I'd suggest you to perform sequential digestion.
Good luck :)
I guess you've already got your answer from the above comments but I would still like to answer your question. Try double digestion first (digestion with both enzymes simultaneously), if this doesn"t give you correct result then do sequential digestion (for example first with SacI and then with BamHI). You will most probably do an agarose gel extraction of your desired band after the first digestion which will remove any traces of the first ezyme's buffer. And your sample is ready for the second digestion.
The buffer composition is different for different manufactures with respect to the digestion enzymes. therefore it is better to use it from the same manufacturer.
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