PstI/SapI digestion problem - restriction enzyme (Sep/04/2007 )

Hey, folks

I've been playing with PstI/SapI for a while...Firstly there was no cleavage, so I changed to fresh SapI enzyme -- remember that SapI expires in a short time (a few months).

Then I found something really like star activity, that after 1.5 hour at 37'C incubation, The DNA was chewed up into small fragments. I supposed to see 2k and 200 bp, but I actually saw 2k, 1k, something 800, 500, weak or unrecognizable 200. The problem may come from the amount of the enzyme used. Like in my 20uL scale of digestion, I used 20 units of PstI and 4 units of SapI (because it is supplied as 2000 units/mL and I used 2 uL). Anyone sees anything wrong with this?

Or it can be the incubation time. Could it be too short (1.5 h) to cut completely?

Or anything you can think of...


Lydia

exotic enzymes... Is it at all possible to use some other restriction enzyme other then SapI?

Well from looking at the number of units of enzyme in use and the volume stated, I would say you may be experience problems with glycerol. From the looks of it 2ul of SapI and 2ul of PstI are being used in a 20ul volume. Glycerol while a great protector is also an inhbitor of enzyme activity. Inhibition and star activity occurs when glycerol concentration reaches and exceeds 5%. 4ul of 20ul is far too high. I would recommend increasing the volume of your digest and reducing the amount of enyzme in use. The amount of enzyme in use can be adjusted by taking in consideration how much DNA is being cut.

Finally as mentioned on the NEB website, ends made by SapI ligate poorly. So please be advised. Again, is it not possible to use some other enzyme?

It is a great point to reduce enzyme volume. Thank you!

But I use SapI for a special purpose. It is going along with the pTWIN1 vector with which I'm trying the intein (self-cleave peptide) strategy to create Cysteine-leading peptide. Seems unavoidable...

are you certain? You can make large primers upto 100bp long.