Problems digesting PCR product - troublesome enzymes: NotI PvuI (Mar/11/2009 )

Hello,
I am attempting to sub-clone a potassium channel into a shuttle vector to make a virus. I am using PCR to put the NotI site on the N terminus of my channel and PvuI on the C terminus. My restriction enzymes are from New England biolabs and according to their website, NotI cuts with 10% efficiency after 2 hours, and PvuI will cut with 10% efficiency after 20 hours. Here is the link to the site where I found this information (both of my primers have 5 nt before the restriction site and approx 20nt after) http://www.neb.com/nebecomm/tech_reference...nucleotides.asp
I am planning on cutting my PCR product with PvuI overnight followed by NotI for two hours. Is there any way to improve my chances of having the same PCR strand cut by both enzymes? Is there any way that I can identify the DNA that has been digested so I only use that in my ligation?
I am relatively new to cloning so I would appreciate any advice you have for me!

Thanks!

vector: 8.9Kb (appears to be cut well by both enzymes)
insert: 2.7Kb
insert after PCR: 5' TTA TT NotI Start Potassium channel Stop PvuI CG CGG 3'

jlolsen40 on Mar 11 2009, 08:49 AM said:

Um.... I would recommend that the primer be redesigned. This problem has the makings of an epic challenge, heroic acts and dogged perseverance in the face of despair.

If the forward and reverse primers should be given longer guards. Rather than 5bp, it should at least be 8bp. Since PvuI is unknown I would go so far to add 10bp of guard sequence. Redesigning the primers would be the better/safer and in my opinion faster/cheaper option.


However if you must.
Both PvuI and NotI work in NEB buffer 3. So conduct an overnight double digest using both enzymes. Use more enzyme to cut your PCR product. However the total volume of enzyme used can not exceed 5% of the digest volume. The enzymes comes in glycerol which preserves the enzymes but also inhibits its activity. So adding too much enzyme has the effect of inhibiting digestion or worst cause star activity.

It is possible that you might end up having to do a blunt end ligation to insert this PCR fragment (with current Primers)

jlolsen40 on Mar 11 2009, 08:49 AM said:

In theory you could have used biotinylated primers. So if the digest worked, the digest PCR product will elute from a streptavidin column.

jlolsen40 on Mar 12 2009, 03:49 AM said:

Adding to what pernesblue said, epic challenges and heroic acts are all very well, but give me a short turnaround successful experiment any day! Go for a longer sequence at the ends; primers are pretty cheap!

A quick test of your digestion is to take some of the reaction, kill the enzymes and do a short (~20 min) room temp ligation, before running it, along with some of the digested PCR product, on a gel. At the same time, try a religation on single-digestion product, as well as DNA ladder, to make sure the ligase is behaving itself. The single-cut DNA should only make dimers, your double-cut DNA should make dimers, trimers and hopefully higher multimers, while the ladder should generally shift up in the weight range.