Restriction enzyme digestion problem - (Aug/05/2010 )
I have tried many times to linearise a 10kB plasmid using NEB pacI. After digesting it for 2hrs at 37C and running a 1% and also tried it with 0.7% Agarose gels I still see a band on the region where the wells are. I can't figure out if the plasmid is digested and linearized or if its supercoiled/uncut cos the bands are too bright in the region of 8kb-10kb. I digest ~5ul of DNA is a total volume of 50ul.
I have not let it digest over night cos I don't want the DNA to be digested too much. Is anyone familiar with the PacI enzyme or use it frequently. It seems that it is a time saver enzyme and need to complete the digestion in 2hrs.
Any advise in order to linearise this fragment is much appreciated since this is the only enzyme that is unique.
Could you post a picture of your gel?
molecule on Fri Aug 6 00:47:15 2010 said:
I have tried many times to linearise a 10kB plasmid using NEB pacI. After digesting it for 2hrs at 37C and running a 1% and also tried it with 0.7% Agarose gels I still see a band on the region where the wells are. I can't figure out if the plasmid is digested and linearized or if its supercoiled/uncut cos the bands are too bright in the region of 8kb-10kb. I digest ~5ul of DNA is a total volume of 50ul.
I have not let it digest over night cos I don't want the DNA to be digested too much. Is anyone familiar with the PacI enzyme or use it frequently. It seems that it is a time saver enzyme and need to complete the digestion in 2hrs.
Any advise in order to linearise this fragment is much appreciated since this is the only enzyme that is unique.
hi
From your post it seems that there is more of the uncut DNA , than the linearised one. What is the concentration of the DNA do you use for restriction digestion. Try this method, it works. Take less than 1 microgram of DNA and add 2 ul of the correct buffer and 0.5 ul of the enzyme and make up the volume to app 15 or 20ul. Leave it for two hrs, dont not take before that. Then run it on the gel, you will get the required bands. Always be sure that large amount of the enz will not work sometimes. Everything should be in the appropriate concentration, only then the restriction digestion works. This is what I have found out with my experience. See if it gets linearised completely, if it is not, next time just increase the time and not the enzyme. If that works then take the required concentration of the DNA you need and do the digestion. Hope this should be fine. In case you need any help on this, you can mail me at ajayaa2020@gmail.com. Good luck