detection of restriction? - (Sep/26/2009 )
i have a 387bp template. its primers have Nde1/EcoR1 restriction sites. i usually keep it for 4hrs incubation at 37C. how can i make sure that my primers have got restricted?
i usually go for ligation followed by transformation. but this is not the right procedure b'coz i don't know the correct incubation time for restriction.
plz suggest me some alternative
4hr at 37C is more than enough time- I usually only do 1 hr and depending on your template quality and amount of DNA vs enzyme, even that is more than enough time. Do you purify your product before digesting it??
As far as I am aware, there is no way to check if your product is properly digested, other than successful ligation and transformation of course. You could check your enzyme activity with another template......but all this will tell you is that your enzyme aliquot is still active- that doesn't mean it is definitely working in your PCR product digest.