restriction digest - smear - (Dec/08/2010 )
Hi everyone, I would like to ask for some help concerning my restriction digest..
Im trying to get a restriction pattern for a 12kbp PCR product, however, all i get is always a smear.
I cleaned up the PCR product and used nuclease free water for elution, checked the DNA concentration (30 ng/ul)and used 10 ul for the digest. RE used was MboII and i incubated at 37 °C for 2 hours, then inactivated the enzyme at 70 °C for 20 min.
Im attaching a picture of my last digest, first lane is lambda DNA (dam-) then 3rd lane is my digest and marker. Im expecting fragments between 1000 bp and 250 bp... What do i do wrong?
Thanks a lot for help, Zuzana
A lane having your undigested product would be helpful. I would suggest that this gel might show degradation of your DNA. It would be important to know whether the degradation happened before or after the digestion. If this is purely a diagnostic digestion, you likely need not purify the PCR product, but could simply add restriction enzymes and incubate the mix prior to running the gel. Again, I would save some of the pcr product without digestion for the gel.