Hello ,
So i digested pBeloBAC11 using HindIII ( left the eppendorfs 1 hour in the water bath for the digestion process ).
After that the sample was frozen at -20 C and next day a gel electrophoresis was run using the samples (image below). The gel was run for 1 hour at 75 V. The gel percentage was 1% . The DNA marker size is 1kb ( I know I should be using a bigger one , but i didn't have any other DNA marker available at that time ).
The problem is , I always get lost with the digestion process and i can't understand if my digestion worked or not. Was 1 hour enough for HindIII to digest pBeloBAC11? Was the gel percentage to high or low ? Should i run the gel for a longer time or at a different voltage ?
It will be helpful if somebody can explain what is going on ....
Thanks a lot , sorry about this !
I believe you are talking about HindIII. pBeloBAC11 has only one HindIII site. I can see a band at very top. you haven't ran the gel for long enough that you can determine its molecular weight but it seems quite high. may be 7500 bp. its hard to tell if the vector is single cut or uncut. what you can do is run uncut and cut gel and compare. supercoiled circular DNA should run differently than linear DNA.
run it longer on 0.75% agarose along with uncut vector single cut should linearize your vector and you might not see it anyway. Run your cloning with digestion control and seee if you have any colonies
First of all I agree with Jaroslaw Krol that you are using too high percentage of agarose, 0.7 or 0.75 is more appropriate and if you run cut and uncut samples side by side you should be able to tell whether the digestion is working well or not.
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