PCR and restriction enzyme digestion - (Feb/16/2013 )
hello, i done transformation on dh5-alpha culture and the culture is grown, when i screen the culture by performing PCR, it give positive result (band shown at expected size). however, when i retest with double digestion, there no band even for the positive control. i used 200 ng and 10U (0.5 ul) at 37oC for an hour then inactivate immediately at 65oC for 20 minute? is it my transformation is really worked? any recommendation?
If your + control didn't work - then you can't say anything about the experiment... you either need to troubleshoot the experiment until your + control works, or find a different way to get the confirmation you need.
do you means i still need redo the transformation,or just optimize the extraction for restriction enzymes digestion?some of my colleague said these happen because of insufficient concentration of DNA that need for the digestion..
I would start with getting the digestion working - that's your test. You have some evidence that your ligation is working from the PCR.
Keep this in mind - a small fragment of DNA cut out of a plasmid may not show up on a gel if there isn't enough DNA to bind the EtBr and give a visible band. So, 200 ng of plasmid may not be enough to see a cut out band, though you should be able to see the initial plasmid band.