HDTenEightyP/NetNews huggingface dataset:
From: ccy@po.CWRU.Edu (Cheung C. Yue)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Synthetic oligo with "blocked" 3'-OH group
Date: 16 Dec 1992 17:06:28 GMT
Organization: Case Western Reserve University, Cleveland, OH (USA)
Reply-To: ccy@po.CWRU.Edu (Cheung C. Yue)
NNTP-Posting-Host: slc12.ins.cwru.edu


Seems like a crazy idea, but the paper on "ligation-anchored PCR" in 
PNAS 89:9823, strongly suggests the utility of such 3'-blocked oligos.
In that paper, the 5'-RACE procedure is now an anchor oligo ligation
procedure.  Briefly, cDNA is primed by a sequence-specific oligo, and
ligated to the anchor oligo which is 5'-phosphorylated and 3'-blocked
(by tailing with dideoxynucleotide) using RNA ligase.  This ligated
species can then be amplified by PCR using sequence-specific oligo and
an oligo complementary to the anchor.  The absence of 5' phosphate and
presence of 3'OH on the sequence-specific oligo, and the reverse for
the anchor oligo ensures a one-way ligation.

Real neat method.  I was just thinking that if in the synthesis of the
anchor oligo, one could chemically knock off the 3'-OH while cleaving
the oligo off the column, then one could avoid the tailing reaction which
is obligatory in the current version of the procedure.  Not being a DNA
synthesis chemist, I don't know how one might do that (without screwing
up the rest of the molecule).  Any suggestions?

ccy@po.cwru.edu
--
From: GIETZ@bldghsc.lan1.umanitoba.ca
Newsgroups: bionet.molbio.methds-reagnts
Subject: RE:Q: fusion protein expressin
Date: 18 Dec 92 07:17:00 GMT
Distribution: bionet

Jackson,
    We have been recently been using the pGEX system with 
great success.  We do use another strain; JM109.We have tried
 other strains without luck (DH5 alpha).   We also do not do the column thing.
 
We just put in 50 to 100 ul of a 50% slurry into a 1 ml extract and batch it. 
 
We have also found that glutathione
only removes about 1/2 of the protein that is stuck, as assayed
by cracking the beads with SDS PAGE loading buffer.  
This sacrifices beads but it is one way to assay if things
 are working in light of the inability of glutathione to
elute all the bound protein.  We have used 15 to 50 mM glutathione
 with little increase % eluted.(Still have about 1/2 stuck to the beads
if crack with SDS loading buffer).  But even with this limitation we  
enough protein to see big fat band on a coomassie stained gel.  One thing
you did not mention was the control.  Do you get GST if you just use
non-inserted vector?  This was the first experiment we did and it works
alot better than any fusion we have tried.  We also notice that if we grow 
the bacteria containing the fusion at 25 or 30 degrees rather than 37 degrees
we get more of the full length protein.  In this case we still do a 2 to 4 
hour 
induction and still manage to get loads of fusion protein.
My guess is that if your column is very large, lets say 5 mls, then your
fusion is probably getting hung up because the glutathione does seem to 
be the best elution agent.  Try the BATCHing trick with a small amount of 
matrix and crack it with SDS buffer.  I'm sure that if you start with GST
and get this working the rest will fall into place.

Dan Gietz
U of Manitoba
Winnipeg, Manitoba    were its -26 degree C outside now!
GIETZ@BLDGHSC.LAN1.UMANITOBA.CA
From: adavies@vax.path.ox.ac.uk (Anthony Davies)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Cleaning electroporation cuvettes
Date: 12 Dec 92 15:05:26 GMT
Followup-To: bionet.molbio.methds-reagnts
Organization: Institute of Virology, Oxford
Nntp-Posting-User: adavies

In article <David_Nunn-101292131312@tsikar1.life.uiuc.edu>, David_Nunn@qms1.life.uiuc.edu (David Nunn) writes:
> Anyone have a good method for cleaning cuvettes for electroporation?
> Especially useful would be methods that remove any trace bits of DNA, yet
> do not take a toll on the lifetime of the cuvette (no flames please, I know
> they are sold for single use, only :^).  

Wash X6 with dH2O
Zap again
Wash X6 with dH20
Wash with EtOH
Store dry.

No carry over of DNA, no degradation of cuvettes.  An excellent method.

Anthony Davies
Institute of Virology
Oxford UK
From: PHILLIPSA@LARS.AFRC.AC.UK (Andy Phillips)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Expression vectors
Date: 15 Dec 92 17:54:00 GMT
Distribution: bionet
Original-To: METHODS@UK.AC.DARESBURY.DLGM

Thanks to all who replied to my query about expression
vectors.
- Andy
From: chend@ucs.orst.edu (Donald Chen - Microbiology)
Newsgroups: bionet.molbio.methds-reagnts
Subject: sequence and/or restriction map of EMBL4
Keywords: EMBL4, sequence, restriction map
Date: 12 Dec 92 03:46:12 GMT
Organization: University Computing Services - OSU
NNTP-Posting-Host: ucs.orst.edu

Hi:

The subject line says it all. I'd like to find a site where
I can download either the sequence and / or the restriction
map for the EMBL4 vector.  If there is a restriction map,
hopefully it might include approximate sizes (based on the 
linear map). I'm hoping to use the one / or more of the 
following restriction enzymes: EcoRI, EcoRV, PstI.

Thanks for any help,

Don Chen
Microbiology
Oregon State University

chend@bionette.cgrb.orst.edu
or
chend@ucs.orst.edu
From: DMEIER@mis.mcw.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: 3' overlaps in PCR
Date: 14 Dec 92 23:04:06 GMT
Distribution: bionet
Organization: Medical College of Wisconsin (Milwaukee, WI)

Hello All PCR-ers,
  Two potential primers have a 3 base overlap at the 3' end (GGC), 
and have Tm's of 50.9 and 51.2 degrees C.  If I remove a G from, the 
forward primer for example, I get Tm's of 46.1 and 51.2 degrees C with 
a 2 base overlap at the 3' end (GG).  Is it worth sacrificing Tm 
compatibility for reducing 3' overlapping?  Incidently, the Tm of the 
target cDNA is 86.8 degrees C.
  Any comments will be appreciated.

  Thanks.

  Dan
  DMEIER@MIS.MCW.EDU
From: fgarbrec@POST.ITS.MCW.EDU (GARBRECHT)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: IgM for affinity chromatography
Date: 19 Dec 92 17:33:22 GMT
Distribution: bionet

On 11 Dec 1992, NGUYENHD wrote:

> Date: 11 Dec 1992 15:14:22 -0500
> From: NGUYENHD <nguyenhd@hermes.bc.edu>
> To: Methods and Reagents <Methods@net.bio.net>
> Subject: IgM for affinity chromatography
> 
> I have purified IgM antibody from my culture media by Sephacryl 300.  I have
> also tried to couple this antibody to CNBr-activated Sepharose 4B.  Antibody
> binds to the beads, but it loses its activiy and leaks with the eluting buffer
> (0.1M Glycine, pH 2.5).  How do I get around these problems?
> 
> Thank you in advance.
> 
> Hung Nguyen
> Biology Dept., Boston College
> NGUYENHD@hermes.bc.edu

If your antibody leaks off after coupling to CNBr activated beads, then your
coupling was not complete and you are washing off unbound antibody.  Wash the
column more after coupling to make sure all unbound material is freed from
the column before you use it.  Also, some antibodies are quite sensitive
to low pH.  Try successiviely higher pH (2.5, 3.0, 3.5, 4.0, 4.5) to see
whether that will work without denaturing the antibody.  You may also have
better results at high pH (10 or 11).
Fred Garbrecht
Medical College of Wisconsin
fgarbrec@post.its.mcw.edu
From: forsburg@vax.path.ox.ac.uk
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Cleaning electroporation cuvettes
Date: 15 Dec 92 08:32:25 GMT
Followup-To: bionet.molbio.methds-reagnts
Organization: Oxford University Molecular Biology Data Centre
Nntp-Posting-User: forsburg

In article <11DEC199208222046@comb1.comb.umd.edu>, belas@comb1.comb.umd.edu (BOB AT COMB) writes:
> In article <David_Nunn-101292131312@tsikar1.life.uiuc.edu>, David_Nunn@qms1.life.uiuc.edu (David Nunn) writes...
>>Anyone have a good method for cleaning cuvettes for electroporation?
>>Especially useful would be methods that remove any trace bits of DNA, yet
>>do not take a toll on the lifetime of the cuvette (no flames please, I know
>>they are sold for single use, only :^).  
>> 
>>David Nunn, Ph.D.
>>Department of Microbiology
>>University of Illinois
>>Urbana, IL 61801
>>(217) 333-6131

We electroporate yeast.  I routinely wash out the cuvettes in a mild 
detergent, rinse them thoroughly, soak them in EtOH (often overnight) and 
then dry them in a sterile hood.  I've been using the same set for a year now, 
and they are just beginning to show some cracks.  I havent seen any sign of 
carryover of cells or DNA in my expts.

susan forsburg
vax.path.ox.ac.uk
Newsgroups: bionet.molbio.methds-reagnts
From: etaylor@ccu.umanitoba.ca (Euan R. Taylor)
Subject: NON_RADIOACTIVE NORTHERNS- personal summary
Organization: University of Manitoba, Winnipeg, Canada
Distribution: us
Date: Wed, 16 Dec 1992 21:37:22 GMT

I just conducted a short informal survey of DNA DIG users- based on this
80% of users think DIG-DNA probes are utterly unreliable.
In our experience there is no difference in reliability between a DNA
target and an RNA target. The bulk of the replies I have received indicate
that DIG-RNA PROBES are much more reliable.
Whilst radiation is expensive and unpleasant we have returned to it rather
than a system which has failed to provide us with any consistent results.
I  would note that we have no problems doing the same exact experiments
with 32p. Furthermore the consistency of signal strengths with DIG is
"pathetic" for instance - if we do a series of DNA dilutions and probe
with DIG-DNA there is no consistent gradient of signal intensities
ie. signal strength is not proportional to target concentration. When the
exact same dilutions are applied to a blot and hybridised to 32p we get a
perfect intensity series (same DNA, same manifold, same membrane, same
tubes, same blot, done in duplicate.)
Now I am not saying that this system "never works" clearly some people
like it, however my impression is that it can be very unreliable, and in our
case has involved a considerable expenditure of time and effort in a
futile attempt to get even the most basic and simple controls to work
reliably. 
Our problems are not related to the blotting side of the procedure because
we can re-probe unsuccessful DIG blots using a 32p probe (made from the
same DNA sample)  and get perfect, reproducible, interpretable results,
with no problem.
I just think that anyone who is thinking of moving over to a
non-radioactive system would be well advised to insist on some samples to
try before they start laying out batches of $500 and days and days of
time, on using the system.

Euan Taylor
etaylor@ccu.umanitoba.ca

The above opinions are my own and are nothing to do with, and do not
necessarily represent those of my institution
From: rpgrant@vax.path.ox.ac.uk
Newsgroups: bionet.molbio.methds-reagnts
Subject: Taq - not so promiscuous???
Date: 14 Dec 92 13:33:12 GMT
Organization: Oxford University Molecular Biology Data Centre
Nntp-Posting-User: rpgrant

Following the recent discussion about various polymerases used in PCR, I'd 
like to add my tuppence.
Recently I amplified, cloned and sequenced two lengths of DNA, 1.35kb and 
1.9kb, using Taq.   To my surprise, I found that the sequence of the first 
clone in both experiments was 100.000% correct.  (BTW, the amplifications also 
incorporated restriction sites and eukaryotic initiation sequences).

Now, was I exceptionally lucky, or was it the little trick my boss suggested? 
Namely, I used 50uM dNTP in the amplification reactions instead of the normal 
200uM.  
It would be interesting to see if this modification increases fidelity in 
general......
BFN,
Richard
From: DMEIER@mis.mcw.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Mobility Shift Assay
Date: 18 Dec 92 21:43:22 GMT
Distribution: bionet
Organization: Medical College of Wisconsin (Milwaukee, WI)

As for as dyes are concerned, don't add any dye to your samples because 
the dye can inhibit protein/DNA binding.  I got around that problem by 
prerunning a dye-containing solution a few centimeters into the gel 
before loading my samples.  Worked well for me.

Dan
DMEIER@mis.mcw.edu
Newsgroups: bionet.molbio.methds-reagnts
From: vjongene@isrec-sun1.unil.ch (Victor Jongeneel)
Subject: Re: DNA from Polyacrylamide & Protein Fusion and Purification System
Organization: University of Lausanne CH (Switzerland)
X-Newsreader: TIN [version 1.1 PL8]
Distribution: bionet
Date: Thu, 17 Dec 1992 07:12:00 GMT

Basavaraju Shankarappa (bsh@MED.PITT.EDU) wrote:
: >   Hi! I was wondering if anyone out there knows of any company that sells a kit
: > for extracting DNA from polyacrylamide gels. According to Molecular Cloning
: > (2nd Edition) by Sambrook et al on page 6.46, there exists a "Crush and Soak"
: > method. However, the method is lengthy and inefficient. With so many kits
: > available for extracting DNA out of agarose gels, I haven't been able to find
: > one that's for use with polyacrylamide gels. Unfortunately, I must run my DNA
: > samples on PAGE. I would appreciate it if anyone could provide any info. or
: > even suggest a better method other than "Cursh and Soak".
: > Michael Cheng
: > Dept. of Biological Chemistry
: > Kyoto University
: > a52041@sakura.kudpc.kyoto-u.ac.jp

: There is a system of solubilizable acrylamide gels for isolation of DNA
: fragments by Dr. Norman Hansen.  He was our teacher and he was a great
: one.
: 	In this system, the gels are crosslinked by disulfide bridges and
: the gels are supposed to be quite effective in resolving MW ranges from
: 21 kb down.  I do not know whether anybody markets this system.  You can
: obtain the disulfide containing bis from Bio-Rad.  The gels are solubilized
: by treating with mercaptoethanol and the DNA purified by DEAE cellulose. 
: I guess you should be able to clean DNA with other methods also.
: 	The reference is J.N. Hansen.  Use of solubilizable acrylamide 
: disulfide gels for isolation of DNA fragments suitable for sequence
: analysis.  Anal. Biochem. 116: 146-151 (1981).

: Raj Shankarappa
: bsh@med.pitt.edu

Michael,

Another (and easier) way is to electro-elute your DNA from the gel.
There are several commercial systems available to do this.  We have
tried the BioTrap from Schleicher & Schuell (may be sold under
different trade names outside Europe) and a "salt trap" model sold by
IBI/Kodak.  They both work fine, but I prefer the IBI system, which is
quicker and allows you to elute up to 6 samples simultaneously.  All
you have to do after elution is EtOH precipitation.

Hope this helps.  Good luck!

Victor

--------------------------------------------------------------------
C. Victor JONGENEEL                       vjongene@isrecmail.unil.ch
Ludwig Institute for Cancer Research, Lausanne Branch
Chemin des Boveresses 155                       FAX: +41-21-653-4474
CH-1066 EPALINGES				Tel: +41-21-653-6275
Switzerland
--------------------------------------------------------------------








Xref: sparky bionet.molbio.methds-reagnts:2893 bionet.general:1981
From: rpgrant@vax.path.ox.ac.uk
Newsgroups: bionet.molbio.methds-reagnts,bionet.general
Subject: ENZO
Date: 14 Dec 92 09:05:35 GMT
Organization: Oxford University Molecular Biology Data Centre
Nntp-Posting-User: barclay

On behalf of a colleague...
Has anyone heard of the company  Enzo ???
They sell biotin 11 dUTP.
I'm sorry, we don't have any other information.
Thanks,
Richard
RPGRANT@vax.path.ox.ac.uk
From: Jorges@bcm.tmc.edu (Jorge L. Sepulveda)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Mobility Shift Assay
Date: 16 Dec 1992 23:57:36 GMT
Organization: Baylor College of Medicine, Houston, Tx
Distribution: bionet
NNTP-Posting-Host: 128.249.21.72

In article <1992Dec15.145242.3047@gserv1.dl.ac.uk> 
LCNAVOT@WEIZMANN.WEIZMANN.AC.IL (Nir Navot) writes:
> I've been trying to perfect the conditions for running a Mobility Shift 
Assay.
> I'm using this assay to measure the DNA-binding activities of various 
proteins.
> The probe is a 20 mer ds piece of DNA with 4 bases extension at each end 
and it
>  is labled by kinasing. I have been runing the reactions in 4% 
acrylamide gels
> in 0.5xTBE, at 50-100 V at 4 C (cold room). The bands I'm getting are 
smeared
> and smilling (though I'm not).
> Could I please have your comments regarding the following points:
> 1. Presence of protruding ends on the labled oligo
> 2. Kinasing vers. filling-in for labaling
> 3. Poly d(I-C) as non-specific competitor vers. sonicated calf-thymus DNA
> 4. The use of 0.5 x TBE as running buffer, and running conditions in 
general
> 5. Your chosen loading buffer
> 6. Any other tricks?
> Thanks in advance!!!
> Nir

Nir,

Here are a few tricks,

1. I always purify my ds oligos, either after annealing or after 
filling-in with Klenow; if you choose the hot nucleotide as the last to be 
filled in, you know that all you labeled probe is double stranded and 
blunt ended (although the incorporations are lower, but I use only 10,000 
to 20,000 cpm/lane)
2. Maybe for the above reason, I find filled in probes to have much less 
background than end-labeled
3. For some probes, 1 g/reaction of E. coli DNA is better, for others I 
use 1 g/l of dIdC
4. I use 1/4 TBE and run it at 10W, 4C
5. I just add dyes (XC+BB) to the sample immediatly before loading, 
because there is enough glycerol to sink the sample
6. Be carefull with the order of addition of reagents; I do buffers, 
competitor dna, nuclear extracts, wait 5-10', add hot probe
7. The amount of glycerol (may be because it interacts with borate) can 
cause a lot of smearing and background; keep it less than 4% in the 
reaction

Cheers, 




Jorge L. Sepulveda, 

Dept. of Pathology,                        Email: Jorges@bcm.tmc.edu
Baylor College of Medicine                 Telephone: 713-798-7330
One Baylor Plaza, Houston, TX 77030        Telefax: 713-798-5838
From: bmcnulty@OAVAX.CSUCHICO.EDU (BOB MCNULTY)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Plant protein isolation
Date: 17 Dec 92 16:28:37 GMT
Reply-To: bmcnulty@OAVAX.CSUCHICO.EDU
Organization: California State University, Chico
NNTP-Posting-Host: oavax.csuchico.edu




	I am working on an extraction of a plasma membrane protein from plant
roots.  After separation in an aqueous phase system (dextran/PEG), I have
not had luck with phenol extraction of the protein from the membrane for
second dimension electrophoresis (the protein precipitates with methanol,
but transfer to the gel is poor).  I am considering n-octyl-B-D-
glucopyranoside, but need suggestions as to concentration, incubation, and
mostly a method to remove the detergent for 2-D electrophoresis.  Due to
funds, columns aren't really an option, and I have an extremely small
membrane pellet, so a series of detergent test ratios is hard to commit to!

Any suggestions would be greatly appreciated.

               Thanks in advance.
        ////////      "Great spirits have always encountered violent opposition
       (       )        from mediocre minds."   
        \ @ @ /                          A. Einstein
-------w---U---w----
Bob McNulty, CA. STATE UNIV., CHICO
BMCNULTY@OAVAX.CSUCHICO.EDU
From: bsh@MED.PITT.EDU (Basavaraju Shankarappa)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Removing RNA from a mixture of protein and RNA
Date: 17 Dec 92 16:10:20 GMT
Distribution: bionet


	Hi folks:

	Sometime back there was a question about removing RNA contamination
from a purified protein solution.  Among the many suggestions that were
provided including precipitating proteins selectively some were neat.

	There is one more way.  US biochemicals sells a large list of
immobilized enzymes and RNase A is one of those.  I think this would be
a good idea if you can purify the ribonucleotides away from the protein
or if it does not matter.
Raj Shankarappa
Univ of Pittsburgh
bsh@med.pitt.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Cleaning electroporation cuvettes
From: cytogen@chmeds.ac.nz (Martin Kennedy)
Date: 15 Dec 92 15:37:21 +1200
Followup-To: bionet.molbio.methds-reagnts

In article <David_Nunn-101292131312@tsikar1.life.uiuc.edu>, David_Nunn@qms1.life.uiuc.edu (David Nunn) writes:
> Anyone have a good method for cleaning cuvettes for electroporation?
> Especially useful would be methods that remove any trace bits of DNA, yet
> do not take a toll on the lifetime of the cuvette (no flames please, I know
> they are sold for single use, only :^).  
> 
> David Nunn, Ph.D.
> Department of Microbiology
> University of Illinois
> Urbana, IL 61801
> (217) 333-6131


David,

I've not had to recycle my EP cuvettes yet, but I have several hundred of them
lying around in case times get tough!  I noted in a product review in Nature
347, page 310 a year or two back that an ABI robotic PCR machine uses a dilute
nitric acid wash to remove DNA from its single PCR plate (I forget whether it
was aluminium or not, but I suspect so).  I reckon that if nitric acid can
clean a plate sufficiently that there is no carry-over of DNA between the PCR
reactions then it ought to be effective at cleansing EP cuvettes.  Perhaps it
is worth a go - let me know if it works!

Cheers,

Martin Kennedy
Newsgroups: bionet.molbio.methds-reagnts
From: horton@molbio.cbs.umn.edu (Robert Horton)
Subject: Re: Taq - not so promiscuous???
Organization: University of Minnesota
X-Newsreader: Tin 1.1 PL5
Date: Tue, 15 Dec 1992 21:03:13 GMT

	Your observation of a low rate of incorporation of errors by Taq
is not unusual. We found no errors in over 6000 bases from clones amplified
from cDNAs (J. Immunol. 145:1782-7, 1990). The reaction conditions were
"standard" except that the [Mg++] was a little low (1mM). We had slightly
higher error frequencies in other experiments, which happened to use slightly
more Mg. (dNTPs were always 200uM each).
	I think Taq often gets a bum rap as far as errors introduced into
clones is concerned. The key may be that error _frequency_ (i.e., the
observed fraction of errors incorporated into the product) is not directly
correlated with error _rate_ (the fraction of wrong bases incorporated
by Taq). This is because of "thermal proofreading" (as I believe they call
it in California), in which DNA strands that get the wrong base added don't
get extended efficiently by polymerase, so these errors don't show up in
the final product.


--
Bob Horton            /\ "Crash programs fail because of the theory that
U. of Minnesota, CBS  || with nine women pregnant you get a baby a month" 
1479 Gortner Ave.    /||\   -Werner von Braun.  Disclaimer:"Bob who?"
St. Paul, MN 55108    ^^   horton@molbio.cbs.umn.edu/(612) 624-3790
From: afc@gnv.ifas.ufl.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: restriction map of EMBL4
Date: 14 Dec 92 11:54:59 -0500

In article <1gbna4INNmho@gaia.ucs.orst.edu>, chend@ucs.orst.edu (Donald Chen - Microbiology) writes:
> Hi:
> 
> The subject line says it all. I'd like to find a site where
> I can download either the sequence and / or the restriction
> map for the EMBL4 vector.  If there is a restriction map,
> hopefully it might include approximate sizes (based on the 
> linear map). I'm hoping to use the one / or more of the 
> following restriction enzymes: EcoRI, EcoRV, PstI.
> 
> Thanks for any help,
> 
> Don Chen
> Microbiology
> Oregon State University
> 
> chend@bionette.cgrb.orst.edu
> or
> chend@ucs.orst.edu

A fairly detailed map is published in Frishauf et al., Methods in Enzymology
volume 153, page 107.  This is a predicted map based on what they used to
assemble the vector, not on data.  (Map is of EMBL 3, so the polylinker is 
reversed).

Andrew Cockburn
USDA