TBE precipitation
XL
use routinely is the TBE. We usually make a 10X stock and make dilution from
there. As many of you may already notice that 10XTBE will precipitate after
the stock is used/opened. I am trying to get some chemistry explanations
about this precipitation. Is it because of the solubility, pH or chemical
reactions with air? Thank you.
--
Xiaoyang Liu
Facility Scientist
Macromolecular Characterization Facility
The University of Connecticut
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Storrs, Connecticut 06269-3149
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Colin Rasmussen
>
> I am running a lot of DNA sequencing gels in my lab. One of the reagents we
> use routinely is the TBE. We usually make a 10X stock and make dilution from
> there. As many of you may already notice that 10XTBE will precipitate after
> the stock is used/opened. I am trying to get some chemistry explanations
> about this precipitation. Is it because of the solubility, pH or chemical
> reactions with air? Thank you.
I think the borate has a tendency to ppt. with time. We found in the
past that autoclaving it helps for some reason. Alternatively, make 5 x
TBE. That always solves the problem. We typically run our gels in 0.5
x TBE so you won't wind up making any more than you are now.
Colin Rasmussen
University of Saskatchewan
Joan Marie Shields
>> I am running a lot of DNA sequencing gels in my lab. One of the reagents we
>> use routinely is the TBE. We usually make a 10X stock and make dilution from
>> there. As many of you may already notice that 10XTBE will precipitate after
>> the stock is used/opened. I am trying to get some chemistry explanations
>> about this precipitation. Is it because of the solubility, pH or chemical
>> reactions with air? Thank you.
Colin Rasmussen <co...@pombe.usask.ca> wrote:
>I think the borate has a tendency to ppt. with time. We found in the
>past that autoclaving it helps for some reason. Alternatively, make 5 x
>TBE. That always solves the problem. We typically run our gels in 0.5
>x TBE so you won't wind up making any more than you are now.
I was having trouble with it precipitating as well - autoclaving seems to
have solved that problem.
I will say regarding the 0.5x - I typically run fairly high percentage
agarose gels (2-4%) and I've found that if I use 0.5x TBE that my bands
aren't nearly as sharp. I've gone back to using 1x TBE and have had much
better results.
joan
--
Joan Shields jshi...@uci.edu http://www.ags.uci.edu/~jshields
University of California - Irvine School of Social Ecology
Department of Environmental Analysis and Design
I do not purchase services or products from unsolicited e-mail advertisements.
Colin Rasmussen
>
> XL wrote:
> >> I am running a lot of DNA sequencing gels in my lab. One of the reagents we
> >> use routinely is the TBE. We usually make a 10X stock and make dilution from
> >> there. As many of you may already notice that 10XTBE will precipitate after
> >> the stock is used/opened. I am trying to get some chemistry explanations
> >> about this precipitation. Is it because of the solubility, pH or chemical
> >> reactions with air? Thank you.
>
> Colin Rasmussen <co...@pombe.usask.ca> wrote:
> >I think the borate has a tendency to ppt. with time. We found in the
> >past that autoclaving it helps for some reason. Alternatively, make 5 x
> >TBE. That always solves the problem. We typically run our gels in 0.5
> >x TBE so you won't wind up making any more than you are now.
>
> I was having trouble with it precipitating as well - autoclaving seems to
> have solved that problem.
>
> I will say regarding the 0.5x - I typically run fairly high percentage
> agarose gels (2-4%) and I've found that if I use 0.5x TBE that my bands
> aren't nearly as sharp. I've gone back to using 1x TBE and have had much
> better results.
>
> joan
What voltage do you run at. We've found that at 100v on a minigel,
sharpness is fine, although I must admit we don't run that high
percentage. When I need to separate smaller things I usually switch to acrylamide.
Colin
Joan Marie Shields
Colin Rasmussen <co...@pombe.usask.ca> wrote:
>> >I think the borate has a tendency to ppt. with time. We found in the
>> >past that autoclaving it helps for some reason. Alternatively, make 5 x
>> >TBE. That always solves the problem. We typically run our gels in 0.5
>> >x TBE so you won't wind up making any more than you are now.
Joan Shields <jshi...@uci.edu> wrote:
>> I was having trouble with it precipitating as well - autoclaving seems to
>> have solved that problem.
>> I will say regarding the 0.5x - I typically run fairly high percentage
>> agarose gels (2-4%) and I've found that if I use 0.5x TBE that my bands
>> aren't nearly as sharp. I've gone back to using 1x TBE and have had much
>> better results.
>What voltage do you run at. We've found that at 100v on a minigel,
>sharpness is fine, although I must admit we don't run that high
>percentage. When I need to separate smaller things I usually switch
>to acrylamide.
Oh, usually between 60 and 90V, depends on the size of the gel. One of
the other fellows here in the lab has also had better results with 1x
as opposed to 0.5x buffer. I think it may depend on a variety of factors.
If 0.5x works for what you are running then stay with it - but if you are
seeing your bands become less sharp than they should be I would suggest
trying 1x TBE.
Ricardo Silva
> I am running a lot of DNA sequencing gels in my lab. One of the reagents we
> use routinely is the TBE. We usually make a 10X stock and make dilution from
> there. As many of you may already notice that 10XTBE will precipitate after
> the stock is used/opened. I am trying to get some chemistry explanations
> about this precipitation. Is it because of the solubility, pH or chemical
> reactions with air? Thank you.
I once read in an old Biotechniques that filtering the TBE stock
through a 0.45 um filter solves the problem. The authors claimed
they could routinely store 50 X stocks!! There was also some
blurbing about dust particles being nucleation centers for TBE.
cheers,
alejandro
Sam Michaelson
Colin Rasmussen wrote:
> XL wrote:
> >
> As many of you may already notice that 10XTBE will precipitate after
> > the stock is used/opened.
>
> I think the borate has a tendency to ppt. with time. We found in the
> past that autoclaving it helps for some reason. Alternatively, make 5 x
> TBE. That always solves the problem.
We routinely make up TBE as 5x, and while it does stay in solution for longer than
10x, eventually it starts to precipitate. BioRad (and possibly other companies)
are offering pre-mixed cask packs of 10xTBE for sale; presumably, they've come up
with some way to prevent precipitation. The autoclaving thing is interesting
though - we never bother because we're only using it to run gels - anybody got any
idea why autoclaving would stave off precipitation?
Sam Michaelson
DSTO Australia
Shaban Javed
Instead of making 10X make 5X it will ppt less quickly
5X TBE
Ameresco Part No J885-4L-R
30175 Solon Ind. Pkwy
Solon, Ohio 44139
1-800-448-4442
Fax no 440-349-1182
also why are you using TBE you can also use Tris Acetate EDTA (TAE)
works just as well.
shaban
--
mailto:shaban...@camb-antibody.co.uk
Shaban Javed
Cambridge Antibody Technology
The Science Park
Melbourn
Royston
Cambridgeshire
SG8 6JJ
England, UK
01763 269262 (tel, Direct)
01763 263233 (tel, Reception)
01763 263413 (fax)
Koen Allosery
Also theBioRad pre-mixed 10X TBE precipitates a while after opening.
Koen Allosery
I have no experienxe with BioRad 10X TBE. So, I do not know if it forms
precipitates.
However, I frequently use the 10X TBE of GibcoBRL (Life Technologies), and
this one definitely forms precipitates a while after opening the box.
Koen.
tfitz...@nexstar.com
>Subject: TBE precipitation
>Date: 12 Aug 1999 10:00:39 -0700
>I am running a lot of DNA sequencing gels in my lab. One of the reagents
we
>use routinely is the TBE. We usually make a 10X stock and make dilution
from
>there. As many of you may already notice that 10XTBE will precipitate
after
>the stock is used/opened. I am trying to get some chemistry explanations
>about this precipitation. Is it because of the solubility, pH or chemical
>reactions with air? Thank you.
ricard...@cigb.edu.cu ("Ricardo Silva") wrote in reply:
>I once read in an old Biotechniques that filtering the TBE stock
>through a 0.45 um filter solves the problem. The authors claimed
>they could routinely store 50 X stocks!! There was also some
>blurbing about dust particles being nucleation centers for TBE.
>alejandro
This is the reference:
Krainer, Adrian R.; Mayeda, Akila. Long-term Storage Of Concentrated
Tris-borate-edta. Benchmark, Biotechniques, Vol.10, No.2, p.182
Article Abstract
LONG-TERM STORAGE OF CONCENTRATED TRIS-BORATE-EDTA
ELECTROPHORESIS BUFFERS WITHOUT PRECIPITATION.
Tim Fitzwater
NeXstar Pharmaceuticals
Joan Marie Shields
>> I will say regarding the 0.5x - I typically run fairly high percentage
>> agarose gels (2-4%) and I've found that if I use 0.5x TBE that my bands
>> aren't nearly as sharp. I've gone back to using 1x TBE and have had much
>> better results.
Jerry M. <J...@843724082793.unil.ch> wrote:
>How do you prepare 4% agarose? I've done 2%, but the thing is always very
>"foamy". How do you avoid having millions of tiny bubbles all over the
>gel? Bunzen burner won't help a lot...
With great difficulty :). They are a royal pain to prepare. I use a
microwave oven and a big flask (to prevent boiling over and into the
oven - messy). A tricky part is knowing how long to let it cool before
adding ethidium bromide and pouring it - it sets very quickly.
It is easier if you use an agarose like nusieve - but it's even more
expensive than the regular agarose.
Junzhi Wei
I would like to transfer a gene into tobacco by Agrobacterium. Could anyone
tell me which company sells binary vector (such as PBI121) or similar
products? Any information or suggestion will be helpful. Thanks.
J.Wei