I digested my cas9 vector by adding 5ug of vector, 3ul of NEB 2.1 buffer, 3ul of BbsI enzyme and completed to 30ul with ultra-pure H2O.
I incubated overnight at 37C and added 1 ul of CIAP incubated for 10 min at 37C.
I ran my digested vector on an agarose gel and there is no visible band.
Although I can see a band for the intact cas9 vector, I don't for the digested one.
I had already made this digestion and I could see a band before, now it disappeared and I tried to make some new digested vector and there is no visible band as well.
I don't understand what is happening, if anyone has any idea.
You might try setting up a control reaction with all the components including buffer, incubating and then running a gel. It may be that your DNA or one of the solutions is contaminated with a nuclease. Especially with an overnight digestion.
Did you really add 5ug of vector? That is a LOT of plasmid for a 30ul reaction.
Thank you for your help! One of my lab team mates used 5ug of plasmid when she used to do cloning and it worked well for her.
But I think it is a lot, how much would you suggest adding?
For the controls, I should add some plasmid with each component individually you mean? Like plasmid + enzyme and plasmid + buffer
I also thought it was maybe the gel extraction kit I was using that was degrading the DNA maybe
did You not see any band before the gel extraction? Or after the gel extraction? That will
tel you if the problem is the digestion or the gel extraction step.
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