I would like to ask around if any of you have done experiment with Restriction Enzyme digestion for short DNA (100 bp or shorter) to get even shorter fragments . Is that possible in your opinions, as I tried several times but I failed to completely digest the DNA (it means the 100bp band still showed up on agarose). I really need some hints for this. Thanks in advance ~
Did you check the restriction sites you fragment has?
Yo can see here: http://nc2.neb.com/NEBcutter2/
If you don´t know your DNA sequence it will be harder!
Sure it is possible, as soon as the reconition site is present, the enzyme will work, the only difficulty will be to control the size of the digested product with the gel electrophoresis, PAG can be the technique of choice in that case.
If you know the sequence of your short DNA fragment, you can find the potential restriction sites and use the enzyme(s) that give you the desired size. You will need to use PAG to detect the products.
Just to extend the digestion time, and the incomplete cuts will disappear.
As Dr. Hermanns said "you should run your fragments on PAGE. Digestion of 100bp for what?
If the restriction site is close to the end of you DNA fragment you need to be sure that you have enough of the overhang of DNA. Each enzyme has different needs in this aspect. Just check specificity for PCR product digestion. You should find it in there. You need to check it for your particular enzyme and beware that enzymes from different suppliers has different needs in this aspect.
I have got a great deal of help from you. Thank you for your kind concerns : ) . I am going to make it following your suggestions ~
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