Doing a double digest (View forum version)


Posted 26 May 2015 - 02:07 PM

I asked something similar in molecular but I think I should ask it here.  I had 150ul of PCR reaction which I used QIAquick-pcr-purification kit on.  I did a 1% agarose gel to check to make sure I had right band before I did the purification.  Now I have 50ul and my next step is the restriction digest with NEB Agel HF and Hindlll HF.  My questions are how of the 50ul should I use and can use both enzymes at the same time?





Posted 27 May 2015 - 12:02 PM

1. You should check the NEB web site for the enzyme. I believe both should use cutsmart buffer, and both can cut at the same time, but you should get into the habbit of checking buffer, temperature, and ability to heat kill.

2. You should cut less than 10 ul of your DNA in a 50 ul reaction. You may get away with cutting more, but it is questionable, because there are often contaminants in your DNA which will inhibit digestion. When the majority of your reaction is watet (which it should be) then you dilute these contaminants.

3. Heat kill your reactions if you can, and avoid unnecessary purification. If you can heat kill, you can go directly to ligation without purification.


Posted 27 May 2015 - 01:02 PM

Thank you, I was going to use 25ul but that makes more sense.  I actually just sent NEB an email about the heat inactivation.  Agel-HF calls for 65 degrees while Hindlll-HF calls for 80 degrees.


The people at NEB are fast, got my answer so will be using the 80 degree for the heat inactivation.


Posted 29 May 2015 - 02:13 AM

if enzym 1 can be heat inactivated at 65°C and the other one at 80°C.... you can just use 80°C of course!

Always use the higher temperature, it will also inactivate the other (at lower temperature) anyway.