NdeI: cutting problems?? anyone else?? seems silly! (fwd)
Neil Harris
---------- Forwarded message ----------
Date: 8 Mar 1996 19:05:24 -0800
From: Heidi Moss <hmm...@MAIL.MED.CORNELL.EDU>
To: "bionet.molbio.methds-reagnts mail newsgroup" <bione...@dl.ac.uk>
Subject: NdeI: cutting problems?? anyone else?? seems silly!
greetings 'netters!!
Has anyone else experienced problems with NdeI (ie. incomplete/non-cutting)?
We have 2 lots from 2 different companies (BM and Gibco) and neither of
them are working. Trobleshooting (ie. altering buffer conditions--there
doesn't seem to be a consensus in the literature) has turned up nothing.
It is a mystery to us and the companies that supplied them. Even on
control DNA!!
What is going on? Is there an NdeI god out there who has cast a spell?
All other enzymes are working perfectly.
If you can shed any light on this problem, please let me know.
thanks in advance!
--Heidi
A colleague of mine told me that NdeI has an extremely short half life
(about quarter of an hour)and she therefore adds extra units during the course of the reaction.
I've had problems using NdeI for cutting plasmids for subcloning i.e.
I've never been able to obtain complete vector digestion and consequently
have to use CIAP even when cutting a polylinker with NdeI and another
enzyme to generate incompatible termini. Usually the second enzyme
works far better than Nde resulting in a high background of self ligated
plasmid.
Hope this tip helps.
Neil Harris,
Plant Science Laboratory,
St. Andrews University.
logan
>Has anyone else experienced problems with NdeI (ie.
>incomplete/non-cutting)?
>We have 2 lots from 2 different companies (BM and Gibco) and neither of
>them are working. Trobleshooting (ie. altering buffer conditions--there
>doesn't seem to be a consensus in the literature) has turned up nothing.
>It is a mystery to us and the companies that supplied them. Even on
>control DNA!!
>If you can shed any light on this problem, please let me know.
>--Heidi
>A colleague of mine told me that NdeI has an extremely short half life
>about quarter of an hour)and she therefore adds extra units during the
>course of the reaction.
>I've had problems using NdeI for cutting plasmids for subcloning i.e.
>I've never been able to obtain complete vector digestion and consequently
>have to use CIAP even when cutting a polylinker with NdeI and another
>enzyme to generate incompatible termini. Usually the second enzyme
>works far better than Nde resulting in a high background of self ligated
>plasmid.
>Hope this tip helps.
>Neil Harris,
>Plant Science Laboratory,
>St. Andrews University.
I concur with the above. In my last project I was using NdeI cut vector for
expression cloning (ATG site) and the person who prepared the vector said
the purity of the plasmid was vital. In the end she prepared her vector by
cesium chloride gradients. She also added NdeI during the course of the
reaction. However, when I prepared my PCR product by gene-clean of agarose
purified band and cut it with NdeI (inserted restriction site in primer) it
cut fine so....? It ain't so simple!?
David C. Logan
*********************************
* david...@plants.ox.ac.uk *
* *
* Department of Plant Sciences *
* University of Oxford *
* South Parks Road *
* Oxford *
* OX1 3RB *
* *
* Tel.(direct): (01865) 275024 *
* *
*********************************
Gary Kucera
present in some DNA preps. For example, DNA purified by standard
miniprep procedures is cleaved at lower rates. In addition, as stated
in your post the half-life is approx. 15 minutes.
Happy cloning,
Gary
Rich Kernan
>
>
>
> >A colleague of mine told me that NdeI has an extremely short half life
> >about quarter of an hour)and she therefore adds extra units during the
> >course of the reaction.
> >I've had problems using NdeI for cutting plasmids for subcloning i.e.
> >I've never been able to obtain complete vector digestion and consequently
> >have to use CIAP even when cutting a polylinker with NdeI and another
> >enzyme to generate incompatible termini. Usually the second enzyme
> >works far better than Nde resulting in a high background of self ligated
> >plasmid.
> >Hope this tip helps.
>
> >Neil Harris,
> >Plant Science Laboratory,
> >St. Andrews University.
>
> I concur with the above. In my last project I was using NdeI cut vector for
> expression cloning (ATG site) and the person who prepared the vector said
> the purity of the plasmid was vital. In the end she prepared her vector by
> cesium chloride gradients. She also added NdeI during the course of the
> reaction. However, when I prepared my PCR product by gene-clean of agarose
> purified band and cut it with NdeI (inserted restriction site in primer) it
> cut fine so....? It ain't so simple!?
>
> *********************************
I have used NdeI extensively in cloning. We buy our enzymes from NEB.
Their catalog says the half life is about 15 minutes, and that is is
sensitive to impurities found in many standard minipreps. We use the
BioRad mini preps which we have found to give VERY clean DNA whan compared
to other brands. I add a little more NdeI than normal (never more than
2.5 ul) and I have never had a problem. Perhaps your DNA is not clean
enough??
--
Rich Kernan
Dept of Microbiology
University of GA
daniel morris
good luck every now and then, use CLEAN DNA, and allow at least six
base-pairs of extra sequence when designing primers containing terminal
NdeI sites.
Its a bastard of an enzyme.
Bernard Murray
dmo...@sbsnov1.auckland.ac.nz says...
Aaargh! I've been reading these posts with a degree of indifference
until this morning when I realised that a probe I've been given
recently needs to be excised with NdeI. I guess the short half life
is inescapable but NEB make no mention about adding BSA. Would this
stabilise the protein or does the enzyme self-destruct during catalysis?
How about extra DTT? Also, can you compensate for dity minipreps by
adding spermidine to the digestion? - this works with other
fastidious enzymes.
Oh for a friendly/non-illegitimate isoschizomer!
Bernard
Bernard Murray, Ph.D.
ber...@elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
Arioch
>
> In article <dmorris-1503...@thermo.sbs.auckland.ac.nz>,
> dmo...@sbsnov1.auckland.ac.nz says...
> >
> >Ditto. Use lots of enzyme, overnight incubations preferably, add more for
> >good luck every now and then, use CLEAN DNA, and allow at least six
> >base-pairs of extra sequence when designing primers containing terminal
> >NdeI sites.
> >
> >Its a bastard of an enzyme.
i dont know what your conditions are, but if possible, dont use NdeI in
double digests. use it individually first and then follow it up with
your second enzyme (if eco or bam, then this method works cuz those 2
will cut just about anything in any condition...). also , the amount of
glycerol in your digest is mucho importante especially if you use a
cranky enzyme like NdeI. check the glycerol concentrations in the
buffer you use and dilute appropriately in your digest. glycerol and
NdeI dont get along too well...cheers...
--
Arioch (s...@topaz.microbio.uab.edu)
Daemon prince of Chaos (a.k.a Cell and Molecular Biology Graduate
student)
The East side of the 9th plane of hell (a.k.a Dept. of Microbiology)
Dimension of Chaos (a.k.a University of Alabama at Birmingham...)
PRETORIUS
>
>Ditto. Use lots of enzyme, overnight incubations preferably, add more for
>good luck every now and then, use CLEAN DNA, and allow at least six
>base-pairs of extra sequence when designing primers containing terminal
>NdeI sites.
>
>Its a bastard of an enzyme.
It also helps if one increases the digestion volume.
Asha
Kiley R. Prilliman
sounds large, but it works fairly well). Use large volumes in
combination with an overnight digest, and it should work well.
Kiley R. Prilliman
Dr. Duncan Clark
<kpri...@rex.re.uokhsc.edu> writes
Overnight won't help when Nde I only has a half life of 15 mins at 37C.
Duncan
--------------------------------------------------------------------------------
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
http://www.dnamp.com
http://www.thenet.co.uk/~dnamp
Jody K. Hirsh
says...
I unfortunately missed the original post. However, if you are having trouble
cutting PCR products after amplification and don't want to buy new primers
with more bases on them, then you can use a Methods in Enzymol.
method--ligate the PCR products together and then digest with the
restriction enzymes and then subclone the products. This will also work
with 1/2 restriction sites of 6-mer palindromic sites.(V. Jung et al.,Meth. in
Enzymol. 218:357-62, 1993).
---
Jody K. Hirsh
Northwestern University, Chicago, IL. USA
jkh...@nwu.edu
Anthony Tomlinson
name stands
for Never Cuts. Just try the usual things, especially overnight digests
with
a small excess (5-10 fold) of enzyme.
Anthony.
gerry oneill
This may be due to the dam methylation status of the DNA sourse; ie
dam +ve plasmid DNA
NdeI cannot restrict dan+ve DNA but its isochizomer Dpni can
gerry
Ashok Aiyar
NdeI recognizes and cuts "CA/TATG". There is neither a dam methylation
site within that sequence, nor is NdeI an isoschizomer of DpnI,
which recognizes and cuts "GAm/TC" (where Am is an N6 methylated dA).
NdeI however has a very short half life and is very sensitive to
impurities in the DNA preparation. These contribute to it's being
characterized as "poor" enzyme.
Ashok
--
Ashok Aiyar
Department of Oncology ai...@ebv.oncology.wisc.edu
Univ. of Wisconsin-Madison tel: (608) 262-6697
Dr. Duncan Clark
<ai...@ebv.oncology.wisc.edu> writes
>On 17 Apr 1996 21:56:24 GMT, gerry oneill <10173...@CompuServe.COM> wrote:
>>Ndei cutting problems
>>
>>This may be due to the dam methylation status of the DNA sourse; ie
>>dam +ve plasmid DNA
>>NdeI cannot restrict dan+ve DNA but its isochizomer Dpni can
>
>NdeI recognizes and cuts "CA/TATG". There is neither a dam methylation
>site within that sequence, nor is NdeI an isoschizomer of DpnI,
>which recognizes and cuts "GAm/TC" (where Am is an N6 methylated dA).
I think he meant Nde II which is also present in the Nde I strain and is
an iso. of Mbo I. As Nde I from NEB is recombinant no Nde II will ever
be present in the purified enzyme prep.