Usually, a uncut plasmid should show at least three bands on gel. The top band most likely is the nicked/linearized plasmid. A mid band is relatively thicker which is the supercoiled plasmid. And if there is a lower band most likely the single circular formation of the plasmid.

So, when you run the digested products on a gel, if the digestion was successful, you will see only one band which should be the same size as the top band of your uncut plasmid.

One suggestion for your digestion setup is that it might be easier to consider using double digestion (in one reaction) than sequential one. Both BamHI and NheI have high-fidelity version (BamHI-HF and NheI-HF) from NEB. All HF version of enzymes from NEB use buffer 4, which will solve the buffer imcompatibility problem you have.

Good luck !

Thank you Kejun for your help but the problem is that the BamHI is fastdigest and NheI is NEB so I have to do a sequential cut. But the main problem is that the first enzyme I used is BamHI which is FastDigest but when I run it on the gel it gave me unexpected bands which means over digestion (6 hours) star activity and (no difference between cut and uncut (45 minutes) low enzyme units than the recommended.

so what I have to do??

Plz increase the amount of enzyme or reduce the dna conc...and keep it for longer time.

First of all, an uncut plasmid in good condtion will give you only two bands, the uncoiled band on top and supercoiled at the bottom. The cut plasmid will only be one band running in-between, and it sometimes has a weird appearance, as if its top side has little "stars" (can't think of a better description). If your enzyme doesn't cut in a few minutes and it overcuts in 6 hours (this sounds a bit weird to me but maybe it's the fastdigest version), well, cut somewhere in-between? I usually cut plasmids for two hours.

Generally, if you have doubts about your enzymes you can split your vector quantity in two, cut in one tube with enzyme A and the other with B and check if they are completely cut - then add the other enzyme. It will take two hours more but it can save you the next two days.

Finally, how good an enzyme cuts is not only a matter of time. It's also a matter of how much DNA you have, how much enzyme, and how clean the reaction is. Good luck.

BamHI is known to have star activities. I am not particularly familiar with Thermo/Fermentos' restriction enzymes. I would suspect the efficiency of the Fastdigest BamHI enzyme in my first instinct. Really hate to recommend you buying another BamHI from NEB. Guess I am spoiled by the convenience of having a NEB cabinet here right next to our lab.

I agree with Phoebe that try lower the DNA input and restrict the digestion time to 1~2hrs. If still doesn't work, maybe it's time to try getting a new BamHI.

thanks Ranjai and Phoebe

Phoebe: yes it's fast digest enzyme and you're right the uncut one gave me only two bands so I'll try to cut in 2 hours as you suggest may be it'll work with me

thanks again for your kindness you're helpful :)

i suggest to check buffers, whether same buffer or different n if different, is there any company providing some sort universal buffer, purification may also be an important issue, best to use qigen kit for plasmid purification

hope its work

good luck

I do agree with Phoebe & Kejun. You should estimate your DNA quantity & quality. Add DNA aacording to enzyme units being used in ur reaction. Do sequential digestion as Nhe and BamH1 has different reaction activity. Your uncut plasmid DNA will have two bands (usually) and cut DNA will have single band after single digestion.

Good luck

Here's a link:

http://www.qiagen.com/literature/BenchGuide/pdf/1017778_BenchGuide_chap_1.pdf#9

In page 15 there is a photo showing the various things you would expect to see.

We use FastDigest Enzymes in our lab. Guess what the Fast in the name stands for: they are really fast! We use about 100 ng of plasmid and digest them for 10 min. 37°C which is enough time to digest that amount of plasmid. Actually the protocol says that the fidelity of the enzyme is so high that it digests 1µg of phage DNA in 5 min.

Concerning the double digestion: we made the observation that even non-Fastdigest enzymes from other manufacturers work usually very well in the FD-buffer, so that we always try the FD buffer before anything else when making double digests.

Anyway, a picture says more than 1000 words... how about a nice gel pic if your problems persist?

Really thank you my colleagues and friends. I did my experiment and it worked el7 because of your recommendations. Thanks to everyone :)

This is the cut results of my vectors

Hi Marwa

Sorry to hear about your digest issues. Hopefully you'll get to the bottom of it soon.

From what I've read so far it's not clear how much DNA you're starting with, and it appears you are digesting for way too long (especially for an enzyme with star activity). I'm also not sure why you are digesting the plasmid, ie is it for confirmation or for cloning.

If it's confirmation you want then what I routinely do is take around 2ug of plasmid, digest it with around 20U enzyme 1 for 20 mins (one coffee break), run it down a column to wash away the old buffer, and digest the eluate with 10-20U enzyme 2 for 20 mins then run this out on a 1% TBE agarose gel for 30 mins at around 80V. I always run uncut alongside my cut plasmid. Further, if I'm worried about my digest then I run out the two single cut controls too.

Alternatively, if your goal is to clone the excised fragment I'd recommend starting wtih 5-10 ug plasmid and digest with 20-60U of each enzyme (but would still digest for the same length of time).

It's worth mentioning a few things:

- you will note that I pay little heed to the definition of what one Unit of enzyme can cut as these values are worked out under defined and optimal conditions and with a particular substrate - in reality the reaction is many times less efficient. What I've quoted above works routinely for me.

- As you are using a 'fast acting' enzyme I would only digest your plasmid for 10 mins (FYI- I've tried this using the NEB HF enzymes and got excellent cleavage in less than 5 mins).

- if you read the NEB info you'll see that both BamHI and NheI have star activity and are ineffective at cutting supercoiled DNA. With long digestion incubations you have a real chance that you will get star activity, but if you shorten it to 10 mins as I suggest then this becomes a non-issue. Also, I see from your gel image that you have 3 bands in your uncut. As Kejun quite rightly pointed out these will represent nicked plasmid DNA which will migrate near your true plasmid size, and two other bands (supercoiled and relaxed circle) which travel unpredictably. The amount of supercoiled DNA you have is minimal in your prep so no need to worry here.

One final suggestion:

FastDigest is the Fermentas equivalent of NEB's HF enzymes that are all tweaked to work in a single buffer and have reduced star activity. I would bet a lot of money that NEB Buffer 4 and the Fermentas FastDigest buffer are interchangable, and since your NEB NheI works in NEB Buffer 4 I'd suggest performing a double digest in this (or in the FastDigest buffer, or both).

Hopefully this is of some use. Good luck - you'll get there.

Al

Hi Marwa,

i think you should run your gel longer, to start with. How much DNA do you use, what kind of gel?

I recommend something different than Alasdair: for diagnostic digests, use 100 - 200 ng (nano!) of the plasmid, cut it with 1 µl of each enzyme in FD buffer in a total volume of 20 µL either combined or each enzyme alone for 15 min at 37°, HEAT INACTIVATE (70° 10 min) and run 5 µL of the 3 digests and an uncut control on a 1-1.5% TAE gel at maximum 5 V/cm electrode distance for 20-30 min.

Regarding you gel:

Since you haven't included a legend I assume DD means double digested. What i can read from it is:

NheI works, you can see that the typical 3 plasmids bands (199 uncut) now form one band. 222DD has a bigger insert than 199 and 195 DD. Exp DD seems to be a different plasmid, it looks as if it is running about 0,5-1 kb higher than the rest, I even think one can see a slight double band.

##Oops, just saw your last post, so if it worked, congratulations and good luck!

Hi,

I think you should change your RE system by FastDigest enzymes from Fermentas

http://www.fermentas.de/index.php?cPath=1

read and see How its easy

best

I´d recommend sequencing of your plasmid to confirm its identity.

Thank you all