Sequential restriction digestion of plasmid - (Feb/21/2009 )

I am trying to digest pET-15b vector using NdeI and BamHI. Sequential digestion is recommended for this so im going to do NdeI then BamHI after. My question is, is it necessary to purify the now linear plasmid after the first digestion or can i just go on and add the second restriction enzyme, having both in their the same time.

My second questions is how do the circular and linear DNA differ on an agarose gel. Thanks

no, you do not need to purify the linear plasmid after the first digest. NdeI like NEB buffer4. And BamHI can work in buffer 4. Thus the sequential digest begins with NdeI. Once the DNA is linearised, add BamHI and continue the digestion.

An uncut preparation of plasmid DNA contains plasmids in several topologies. cut linear DNA, supercoiled circular DNA and nicked open circular DNA. Supercoiled DNA moves faster than linear DNA as it is more compact. Then comes cut linear DNA (due to shearing). And the slowest of all is the nicked open circular DNA, which has lost its twist and is now a floppy loop. A big circle has greater difficulties migrating through the agarose mesh.

Thus circular DNA doesn't run like linear DNA. And as the DNA ladders are linear DNA, you don't get the expected size.

If you are unfamiliar of how circular DNA looks, I suggest a sample of uncut DNA be run as control.

http://humgen.wustl.edu/hdk_lab_manual/image/plsmid3.gif

shimshady on Feb 21 2009, 10:32 PM said:

Inaddition to pernseblue's suggestion, remember not to exceed the glycerol percentage after adding the second enzyme.

Hi,
It should be ok to perform double digestion at the same time in a single tube.
If the buffer used are not compatible, digest with the first RE with low salt buffer in a small volume, then the second RE with high salt buffer in a increased volume. No need of purification or extraction in between.

Hi,

I am cutting my pET22b vector with Nde1 and BamH1 as well. What I would recommend is sequential digestion too. However, it would be advisable to do a PCR cleanup in between. BamH1 can work in Buffer 4 but its activity is best with Buffer 3. Plus, BamH1 has the problem of STAR activity and you need to remember to add BSA into the reaction to let BamH1 work.

I tried double digestion before, but it didn't work out as well. Hope this helps.

And, I am sorry for this additional question. But how long do you digest the plasmid? I do a 1.5hr digestion with Nde1 first and then a 1.5hr digestion with BamH1. Sometimes, I do a 2hr digestion during the sequential digestion.

The only problem I have is that a full digestion is not ensured and I do get self-religated vector at times. I am thinking of doing an overnight digestion, but this is not recommended for double digestion. Anybody has any ideas?

I do overnight digest (double digest or otherwise) just fine. I think it might be due to the amount of enzyme I am using (very little) in a large volume.

x volume DNA
y volume ds water
5ul of 100x BSA
1ul enzyme
10ul of 10x NEB bufer

total volume 100ul.

To prevent religated vector you should Cip or Sap treat the vector. After your digestions are done, just add one ul of Cip/Sap enzyme to the buffer already present. Incubate at 37 degree for one hour and continue with purification and ligation. This removes the 5' phosphate which is critical for ligation. Now, the vector can only ligate with the insert since the insert has a 5' phosphate after digestion.

perneseblue on Feb 22 2009, 06:31 PM said:

would it be possible for me to have a total volume of 50ul instead of 100ul? like 0.5ul BSA, 1ul Nde1, 1ul BamH1, 5ul NE Buffer 3, plus the DNA and water? I am asking this with regards to overnight digestion.

rkay447 on Feb 22 2009, 09:00 PM said:

I thought of using Cip or Sap, but my supervisor does not provide me with these two. What is the difference between Cip or Sap?

Hey

Calf alkaline phosphatase and shrimp alkaline phosphatase, SAP does work better but has problems with buffer excahnge and involves extra steps, CIP is easy to use as it works in all NEB buffers, so just add, incubate and run on gel.

When it come to BamHI, I always go for sequential digestions, unless its a check digestion.
TC