NEB Restriction Enzymes and Buffer - Restriction (Aug/28/2009 )

Hello,
(this is my first post here... I guess of a long series !)

We have had few unsuccessful restriction in our lab (NEB enzymes) and I am trying to figure out the reason.

I would like to solve 2 doubts:
1) I heard rumors about a change on NEB enzymes formulation in order to make all them compatible with buffer #4, but I do not find this news reported anywhere. Did anyone hear anything about that?

2) I have always stored buffers in the fridge (4ºC) instead that at -20ºC as I now read it is advised in the NEB documentation. I am wondering which is the reason why the buffers should be stored at -20ºC and if storing them at 4ºC could compromise them.

Thanks for your help and discussion!

1) NEB's new HiFi restriction enzymes are the ones that have been modified to work in buffer 4- so if you don't have those, then stick to the usual recommended buffers.

2) I keep my buffers at -20C, but I can't see that 4C would be a problem, I don't know.


Could you give us the details of the digests that aren't working, like what enzyme you are using, what you are trying to digest etc? It could be due to your protocol rather than the enzyme....or something else that someone has experienced also??

Hi
There are a few enzymes that are methylation sensitive.
You should check this each time You plan the restriction.
Good Luck
Michael

Hello bac -- welcome to the BioForums!

Can you tell us which restriction enzymes you've had trouble with? Also, what do you mean by "unsuccessful" -- complete lack of digestion, incomplete digestion, or the production of a smear? What type of DNA are you digesting (chromosomal or plasmid), and how did you isolate it? What is your retriction digest protocol?

We could probably help you out a bit more if we had that information. In general, NEB's restriction enzymes are very good...

Hello,
thank you all for your answers. Here you have more details...

I had 2 *unsuccessful* batches of ligations preparative for cloning.
In both cases:
- I wanted to double digest plasmids propagated in Top10 EColi cells, extracted by Sigma GenElute™ Plasmid Miniprep Kit (PLN350), eluted in di sterile water
- volume of reactions were > or = 30ul and volume of enzyme ~5% to avoid an high concentration of glycerol
- incubation at 37C, 300rpm for 2-3 hours
Result:
- one of the enzymes failed to cut

1st batch) the enzyme that was not working was XbaI in double digestion with BglII in buffer 2, The two restriction sites are very close in the plasmid, so I could see that the plasmid was at least linearized, but single restriction with XbaI were completely uncut, while single restrictions with BglII were completely cut.

To answer Michael: I know XbaI is sensitive to dam methylation and Top10 Ecoli is dam+, but I checked the sequences of the vector and the XbaI restriction site does not carry the dam recognition site. I should then be safe, right?


2nd batch) I then redesigned my cloning plan in order to use 2 different enzymes: SalI-HF and KpnI. Double digestion in buffer 2 again. This time is KpnI that does not cut, same controls as above showed that there is no problem with SalI-HF


It is very probable that the problems were related to the batch of enzymes: unfortunately we are many persons sharing the same chemicals and it is difficult to troubleshoot and track back problems. Furthermore some students have just started to be trained to molecular biology techniques and "Must do/Must not do". But anyway I thought it was better to review everything and make sure I am not making any super-silly mistake. In fact in my previous lab I was used to keep buffers at -20, now I run many more restrictions and found handy avoid to thaw the buffers every time, now I would like to be sure it is safe.

I just found information about the simplification that NEB is accomplishing of the buffer system:
http://www.neb.com/nebecomm/tech_reference...er4_enzymes.asp
I was wondering if it was possible I used an enzyme with "new formulation" with the wrong buffer, (since I was using an old buffer chart to design the restrictions). But, as leelee was pointing out, NEB changed very smartly the denomination of the enzymes whose formulation was changed by adding HF (high fidelity), so this should not be the matter. I used only one HF enzyme: SalI-HF and this one worked nicely. Am I missing any point?


For the moment I bought a new tube of KpnI and will try again the restriction on Monday. If possible I will also use a new tube of buffer. I will let you know!
HomeBrew! Thank you for the welcome, I tried to go through all the main point, but if I missed any, just ask
Thank you again
nice place here !

How much DNA are you trying to cut iin these reactions?
What fraction of the 30 ul volume represents the solution containing DNA?
How was the DNA prepared and purified?

I don't ever recall shaking a restriction digest. No harm, I suppose, but little purpose.

You may eventually have problems with short overhangs if the cut sites are too close, but this does not explain the lack of single enzyme cutting.

Hi
From our experience in the lab, often the troubles are related to the plasmid itself.
Maybe You should sequence the region of interest in your plasmid to make sure that what You expect is really what it is.
Furthermore, Dam recognition site should be checked on both strands.
Hope it is helpful
Best
Michael

bac on Aug 29 2009, 11:02 PM said:

Was your transformation into top 10 a recovery transformation of previously sequenced plasmid or was it from a ligation?
I agree with michaelro, if you transformed a ligation you may just have the wrong thing.

are the sites that worked both contained in the vector/ part with selectable marker gene? (if from a transformation)

edit: how close is close?

Hi there,
thank you very much for all your precious inputs.

@Michaelro, the XbaI site was
ACTAGT TCTAGA GCGGCCGCCAC
and yes: the 2 vectors I wanted to cut SalI-HF/KpnI and use as backbone had been both sequenced and were fine.

Concerning the second restriction KpnI/SalI-HF
Using a new tube of KpnI seemed to work beautifully for the plasmid carrying the insert that got cut out and was then gel-purified. The vector backbone got at least linearized (and was then column-purified), although my control ligations with only backbone cut SalI-HF/KpnI and no insert added gave lot of colonies. I am PCRing few colonies with primers targeting at the edges of the insert to see if I get any good clone.

@lumptal