I was going to digest my plasmids to make a new construct. When I saw the reagent to stop the restriction enzyme reaction, it was undissolved. The reagent consists of 5% SDS in 0.1 M EDTA solution. Another SDS reagent, 0.2M NaCl/1% SDS or popular as Solution II (for DNA isolation), was also undissolved. I decided to read the Sambrook book and then to think what should I do. I searched for the “enzyme digestion” and “plasmid DNA isolation”. I found that the reagent to stop restriction enzyme reaction written in the book is only “0.5 M EDTA” without SDS. Also, the book says that “solution II has to be prepared freshly.” I was just wondering so I decided to ask my sensei.
First of all, I asked about both reagents by showing the tubes to sensei. “Sensei, I think I can not use these reagents and have to make the new ones,” I said. Surprisingly, sensei answered:” you can use these. It always happen in the winter. Just heat it in the hot water,” he answered plainly. I asked the next question because the book said that solution II must be prepared freshly. But, sensei replied me:”that’s not true!” I just smiled and smiled again during our discussion. Yes, it is true that “do not study something only by the book. but, study from the teacher!”
What next? I asked about restriction enzyme stopper reagent. “Why do you use SDS/EDTA, not only EDTA?” He replied:”I like SDS/EDTA very much.” Hoho..what’s that means! He said that when he was young, he used not very pure restriction enzyme, so that the protein can be sticked to the DNA. He needed SDS to denature the protein..EDTA chelats the magnesium ions (or other ions) so that the restriction enzyme stops working. SDS will help to denature the protein so the protein centents in the reaction will be totally denaturated. Moreover, it will be easier to isolate the DNA from the solution! I asked again whether he prepared the enzyme by himself..hehe..”I am not so old. I run molecular biology works in 80-90. People in 80>, they prepared the enzymes theirselves.”
Anything else? (just wait because my memory is not good!)
I had found by the book that 1 unit of restriction enzyme defined as amount required to digest 1 ug of DNA to complete in 1 hour in the recommended buffer and at recommended temperature in a 20 ul reaction. Why does sensei usually use 30 ul of total reaction? Per microlitre of enzyme solution contains 10/15 unit. We usually use 1 ul for only 1 ug of DNA. Is that too much? He answered:”Th unit was counted base on digestion of lambda phage, that’s linear. So we need more enzyme to cut circular plasmid. For the preparation of vector, I recommend you to use 10 times more (it means 10 unit for 1 ug DNA) because we have to digest the plasmid completely. Otherwise, it will produce high background…Enzyme stock from the manufacturer contains glycerol to stabilize at the storage temperature (-20C), so we need to add water at least 10-20 times of enzyme volume that we use (if we use 1 ul of enzyme, we have use total reaction of at least 10 ul. He uses 30 ul so that it will be easier to take the solution when he isolates the fragments.”
Yeah, I completely understand! I am lucky because I did not throw away the reagents and made the new ones. Alhamdulillah.