Hi,
I used BsiWi to digest at 55oC for ~4h, but get smears on my agarose gel. It looks like BsiWi didn't properly cut my plasmids. Since heat-inactivation is at 65oC, I was wondering how crucial the temperature is using BsiWi (I used a water bath and would switch to a PCR machine to be more accurate..). Has anyone experienced problems using BsiWi, and maybe want to share suggestions in terms of digestion time etc.?
Thanks,
Patrick
From which company is your BsiWI?
Some companies still sell enzymes that have star activity, I believe NEB is one of them.
Personally I prefer to perform digestions by using a water bath (because they provide an even heat distribution) and then I would only use the PCR machine to inactive the enzyme.
If you don't have a water bath at 55oC you can improvise one by using a heat block.
Take a 1.5mL tube filled with water and place your digestion in a 0.6mL tube inside the mini water bath.
Can you use BsiWI from Thermo? This version works at 37oC and I never had any problem with DNA degradation using their enzymes.
Thanks for the answer. Andre. I am using NEB-BsiWi, but they claim no star activity even after
Hi Patrick,
If It is from Thermo, I think it will work at 370C. And you can try it for only 3 hours long to digest. Expecting that you have checked the quality of Plasmid by Gel run instead of your NanoDrop Purity. I am using RE for only 3 hours and it's getting me good result in agarose gel. Depending on your smear you can have an RNase treatment with your template.
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