Overview

DNAEASY QIAGEN EXPIRATION
One person found that 6-yr old dnaeasy gave dna that could be PCRed. Several 
people said all reagents but proteinase K would probably be OK.

LARGE AIR BUBBLES IN SEQUENCING GEL
Most suggested removing air in the gel using a vacuum; cleaning plate well;
ˇ°knockingˇ± plate with hand to remove bubbles; putting something small btwn 
plates to ˇ°fish outˇ± the bubbles.  The vacuum is applied before adding APS, 
temed. I was doing experiments to avoid large air bubbles: putting small glass 
plate in 100 gm NaOH per 1 L dd h20 for ca. 5 days resulted in no large air 
bubbles in two gels so far.  I use this same NaOH solution to remove 
AgNO3-stained acrylamide gel from the small plate.

EMAIL RESPONSES
 (I omitted a few responses because most were redundant)

---------- Forwarded message ----------
Date: Sun, 15 Oct 2006 08:17:54 -0700
From: Ward Watt <wbwatt@stanford.edu
To: Ann Eileen Miller Baker <mouse@lamar.colostate.edu
Subject: Bubbles in gels

Dear Ann,

Can't help much with your question about the lifespan of DNeasy
components, except to say that the spin columns themselves are pretty
long-lived.

But as for the bubbles, have you been degassing your gel mix before
adding catalyst? (I assume this is a sequencing or fragment-analysis
gel?) What we do is to put a Nalgene plastic filter unit, 0.2-micron,
on the standard building vacuum line (with a "trap" in between, of
course) and filter the gel mix, pre-catalyst, through into the
collection flask, then leave it under vacuum there for ten minutes
before adding catalyst, gently swirling to mix, and then pouring the
gel. The only bubbles that ever result then are from pouring/sliding
plates too fast (we use an "OWL" casting rack rather than the insane
ABI syringe-and-gaskets rig) or from incompletely cleaned plates.

Cheers,

Ward

Ward Watt
Prof. of Biology

Dept. of Biological Sciences
371 Serra Mall
Stanford University
Stanford, CA USA 94305-5020
Phone (650)-723-4297
FAX (650)-723-6132
Email wbwatt@stanford.edu

Summer --
Rocky Mountain Biological Laboratory
8000 County Road #317
(or P.O. Box 519)
Crested Butte, CO USA 81224
Phone and FAX (970)-349-5668
Same email

A,

 Weak acid = 100 mL glacial acetic with 900 mL dd h20? OR 10 mL glacial
 acetic with 990 mL dd h20? The first step after electrophoresis before
 putting into AgNO3 9no 32P) is to "fix" in 100 mL glacial acetic with 900 mL
 ddh20; my gel stays on the small plate until after I scored.

I would guess the 10% solution would be better, but have no personal
experience. In my grad school lab we used 1N HCl to clean mineral deposits
from glassware.

 I use "glue" (ca. 30-letter name in Sigma catalog) on the shortplate;
 on the longplate, I now use a nonhazmat from Amresco (forgot name), but
 with Westneat we used "sigmacote".

Sigmacote is the one I've used.

Let me know what ends up working for you. Lucky me, I'm on to capillary
sequencing now!

Brian

 ===== Original Message From Brian Ort <brianort11@msn.com =====
 Hi,
 I think acid would work better. I have etched glass as a sort of art 
project
 and I think the etching paste is basic. You definitely don't want to etch
 your plates! Weak acids are very good at removing calcium and other hard
 water deposits (think of acid rain on the Acropolis in Athens or vinegar in
 your tea kettle). I would start with weak acid and don't leave it on the
 plates for more than a minute to start. Rinse well with di-water and wipe
 dry with soft paper towels. Then air dry for a day or so.

 One other thing I've used is a silica coating on the inside of one plate.
 That was to facilitate prying the plates apart after running so that the 
gel
 could be blotted onto filter paper, dried, and used for autoradiography.
 However, I think the silica coating also incidentally helped with pouring
 the gel.

 Cheers,
 Brian

 Brian,
 Do you think NaOH (100 gm per 1 L dd h20)could be effective substitutee 
for
 acid? I did lotsa tapping, but it was useless (so many large bubbles).
 Respectfully,
 A who never degassed acrylamide, but will; who routinely degassed
 starch gels

 ===== Original Message From Brian Ort <brianort11@msn.com =====
 Hi,
 I assume you're talking about bubbles formed while pouring a slab gel
 between glass plates, as for sequencing or microsats. First, you should
 degas the gel solution under vacuum. You're probably already doing this
 since most protocols call for it. While pouring the gel, I've found it
 useful to, first, have the plates lying horizontally on some padded
 support,
 then use my knuckles to tap on the plate at the gel front while I'm
 injecting the gel with a large syringe at the notched end of the plates.
 Tapping causes the gel to advance past any rough spots inside the plates.
 If
 a lab partner is available, it's easier to have one person pour and the
 other tap. So that's a primitive technique.

 I have seen but not used a system made by Owl that supposedly works very
 well for pouring large slab gels. Again, the plates are held horizontally
 and gel solution injected between them.

 Of course, before you begin your plates must be immaculately clean. Any
 mineral deposits on the glass surface will cause the gel solution to
 spread
 around the deposit. If you keep getting bubbles in the same place, that
 might be the problem. A dilute (1N? 0.5N?) solution of HCl can dissolve
 mineral deposits but I don't know what effect it might have on gel
 pouring.

 Best,
 Brian

 --
 Brian Ort
 Postdoctoral Researcher
 Romberg Tiburon Center for Environmental Studies
 San Francisco State University
 3152 Paradise Dr.
 Tiburon CA 94920
 (415)338-3755

From: Puente Molins Marta <martapuente@gmail.com
To: mouse <mouse@lamar.colostate.edu
Subject: Re: Other: (1) dnaeasy qiagen expiration; (2) large air bubbles in
acrylamide gels

Hallo Ann!!!
This is good to clean the plates and remove all the rest of the gel.
But, everytime before start with a new gel you should clean as well the
plates. I can tell you how I use to do...
1) Clean both plates 4 times with water (destilated) and 4 times with Et-OH
(95%). Carefuly, if not the bubbles will appear! (and we don't want this ;-)
).
2) Treat the outher plate (that one in which we want the gel to be fixed,
the smaller one, no?), with the product you usually use for the gel to be
fixed (I use Bind-silan) and after 5 minutes clean gently with Et-OH again,
once.
3) the other plate you'll treat with repellent (I use sigmacote) and after
five minutes we clean again the plate with a dry tissue
4) Build the unit and continue ;-)
Good luck!!! I hope I was useful.
All the best,

Marta
P.D: Please, let me know if everything works!
-- Marta Puente Molins
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK)
Dept. Cytogenetics and Genome Analysis
Apomixis Research Group
Corrensstr. 3, 06466 Gatersleben, Germany
Tel. +49 (0) 39482 5673

Apomixis Group Webpage: http://www.ipk-gatersleben.de/en/02/04/05/index.html

Date: Mon, 16 Oct 2006 09:26:40 -0400
From: Robert E. Marra <Robert.Marra@po.state.ct.us
To: mouse@lamar.ColoState.EDU
Subject: FW: Other: (1) dnaeasy qiagen expiration; (2) large air bubbles in
acrylamide gels

Dear Ann,

I'm looking forward to seeing the answers to your first question (I guess
there's no point in asking Qiagen that!).

Having done thousands of denaturing acrylamide gels, I may be able to help
you with question (2). First, I think it best to always use 1X TBE when
running denaturing or any other kind of acrylamide gel. In fact, I've never
seen a protocol that called for 0.5X. But that wouldn't explain the
bubbles. As far as the bubbles go, I wonder if you're getting these bubbles
in the solution during pouring, or if they're appearing after
polymerization? Some things to check are:
- make sure your APS is of fairly recent vintage.
- Be sure to degas. Some people say they don't, but dissolved
oxygen can interfere with polymerization, so it's a worthwhile step.
- This may be one you have no control over, but I highly recommend
the Bio-Rad Sequin-Gen system. It's a very easy and quick way to pour a
gel, horizontally, via a syringe at the bottom. No tape, no clamps, very
very few mistakes, and we never had problems with bubbles.

Hope this helps,

Bob

Robert E. Marra, Ph.D.
Plant Pathology & Ecology
The Connecticut Agricultural Experiment Station
123 Huntington St
New Haven CT 06511
phone: 203.974.8508
fax: 203.974.8502

Date: Mon, 16 Oct 2006 08:54:30 -0500
From: Kevin J. Roe <kjroe@iastate.edu
To: mouse@lamar.ColoState.EDU
Subject: Re: Other: (1) dnaeasy qiagen expiration; (2) large air bubbles in
acrylamide gels

Regarding the Qiagen kits I would not use any reagents that showed
signs of precipitation in the buffer solutions, otherwise they should
be ok. The proteinase probably does have a shelf life and that would
be my biggest concern. Of course you could buy new proteinase from
another source.

As far the gels, if you are casting acrylamide gels, technique is a
big part of avoiding bubbles. I used to cast large sequencing gels
using 2 55 cc syringes and found that slow and steady worked best.

Hope this helps,

Kevin

From: fishaman@isu.edu
To: mouse@lamar.ColoState.EDU
Subject: Air bubbles in gels

Hi Ann,?br 
I saw your question on evoldir-??br 
I'm not sure if this will help, but when I get bubbles while pouring the gels 
I take a paper-clip or a small spatula and push the bubble to the side of the 
rig. Then it either pops or I can push it up and out of the gel.?br 
Good luck!?br 
Amanda Fisher

Amanda Fisher
Dept of Biology
Idaho State University

Eddy Brede <brede@mpil-ploen.mpg.de =====
Hi Ann,

I regard to your first question, all I would say is try and find out as
depending on how the reagents/columns were kept this could vary!

For your second question I can be a little more helpful. In a previous
lab we used to 'degas' the acrylamide (with a vacuum pump and conical
flask...it will bubble, and then eventually settle) for a few minutes
before adding the TEMED and APS.

Have fun,

Eddie

mb rew <marybethrew@yahoo.com
To: mouse@lamar.ColoState.EDU
Subject: qiagen and acrylamide gels

Hi Ann,
1- I don't know how many years you can keep the Qiagen kits, but I know
that the most sensitive of the components in my experience
is the proteinase K; and although the kit requests that it be stored
at room temperature, I have kept it in a 4 degree fridge and had
fewer problems with it giving out... Just a tip.

2- As for the acrylamide gels, it would really depend on how
you are pouring your gels- there are lots of different rigs
out there for that. If you are using an injection system (which
happens to be what I am most familiar with), then the main problem
usually has to do with having a tiny amount of air in the tip
of your syringe before you start to inject- make sure that is not
the case. Also, many people inject very slowly while knocking
on the glass plates, which I found to be problematic, and opted
for a quicker injection- I ended up with fewer bubbles. And one
other thing is to make sure your glass plates are EXTREMELY
clean, since any tiny bit of dust will give you a bubble. I
always washed my plates just before I was about to pour a gel,
using 1% Alconox soap, and very hot water, then rinse in ddH2O
or as pure water as you have available. Then I would either air
dry them on a rack with the gel side NOT against the rack, and the
the laser end up (so the water drains away from that area) or I
would use one large kimwipe to dry- these tended to leave
less lint/dust on the plate than the small kimwipes, and you
don't have to wait and hour for the plate to dry.

I hope some of this helps!
Mary Beth Rew

===== Original Message From Velavan <velavanp@yahoo.com =====
(1) how many yrs after dnaeasy kits expire are the components still
effective in purifying genomic dna? evidence that genomic dna remains
stable?

can be used upto 1 year and half ..(make sure the buffers are not contaminated
as well as make sure that the lysis buffer is not salt precipitated)

(2) how can I stop getting large air bubbles in gels (6% bis: acrylamide=
formamide-urea-0.5X TBE)?

to get rid of air bubbles i would suggest

Degauze (under vacum filter your gel in liquid form and allow it in vaccum for
20 min) your gel components before you add it on to plate..
to tap the gel plates near to bubbles once gel enters.
the effective way is to clean the gel plates hard to remove acrylamide
particles left already by previous runs.
mostly after preruns small bubbles are gone..

Hope this helps..

Velavan

VELAVAN T P
Animal Evolutionary Ecology,
Zoological Institute,Faculty of biology,
University Tuebingen
Auf der Morgenstelle 28 E
72076 Tuebingen
Germany

Office:+49-7071-2974841
Residence:+49-7071-964735
Mobile:+49-176-24199950
Fax: +49-7071-295634

e-mail: velavanp@yahoo.com,velavan@uni-tuebingen.de
http://www.uni-tuebingen.de/evoeco/
www.velavan.info

"Greater the gifts Greater the Responsibilities"

Ishminder Mann <IMANN@DAL.CA
To: mouse@lamar.ColoState.EDU
Subject: (1) dnaeasy qiagen expiration; (2) large air bubbles in acrylamide
gels

Hi Ann,

I have been using the dnaeasy kits, but not the expired ones. The reagent to
which we add alcohol is most unstable one and you can order that separatly.

For bubbles on gel try to super clean the glass plates, mainly its is dirt 
which
creats bubbles. Also filter you solution (without APS and TEMEED) before
pouring the gel, that really worked for me.

---------- Forwarded message ----------
Date: Mon, 16 Oct 2006 15:14:26 -0500
From: Meredith Mahoney <mjmahoney@museum.state.il.us
To: Ann Eileen Miller Baker <mouse@lamar.ColoState.EDU
Subject: Re: Other: (1) dnaeasy qiagen expiration; (2) large air bubbles in
acrylamide gels

Hi Ann Eileen ,
Qiagen only will guarantee the buffers in the kits for a year. I bet
they work beyond that, but the chemicals probably do degrade over
time. If you don't know how old your kit is, you can call them to
find out by telling them the Lot Number (I recently had to do this).
In addition, the silica filters in the spin columns are coated with a
chemical that helps bind DNA. Again, Qiagen will only guarantee them
for a year, and it seems likely that if the chemical degrades then
the filters will potentially not bind DNA as well as in a new kit. I
don't know how long the kits actually do remain good. My kits were
4-6 years old so I decided not to risk using them.

It's been a while since I poured an acrylamide gel, but make sure
there are no dust specs or anything between the plates. Inject the
acrylamide slowly and steadily. One thing we used to do was tap the
glass at the leading edge of the buffer which helped keep bubbles
from forming (somehow).

good luck,
Meredith

Thanks in advance for your help with two questions; if anyone requests,
I will post your answers/contact info on the evolution net.

(1) how many yrs after dnaeasy kits expire are the components still
effective in purifying genomic dna? evidence that genomic dna remains
stable?

(2) how can I stop getting large air bubbles in gels (6% bis: acrylamide=
formamide-urea-0.5X TBE)?

Ann Eileen Miller Baker

Ann Eileen Miller Baker <mouse@lamar.ColoState.EDU

-- Meredith J. Mahoney
Assistant Curator of Zoology

mjmahoney@museum.state.il.us

Illinois State Museum
Research and Collections Center
1011 E. Ash St
Springfield, IL 62703
ph. 217-785-4843
fax 217-785-2857

Sara Amelia Sandstedt <sandsted@umich.edu
To: mouse@lamar.ColoState.EDU
Subject: bubbles in gels

I used acrylamide gels for
sequencing and for westerns. It depends on your gel setup. If it is a
small gel, and sealed all around, you can pour with the gel at an angle,
with a bottom point down. If it's a thick gel, you might be able to
remove bubbles by sweeping them out with a thin piece of plastic, I'm not
sure.

Make sure your plates are very clean. Never touch the side of the plate
that touches the gel with your hands, only with a piece of soft foam.

Do you use a syringe to pour to gel? If so, you want to pool the liquid
at the top, not inject it. Don't stop pouring and start again. Big gels
that are sealed all around can be tapped with your knuckles as the gel
goes in at an angle. They can also be lifted to different angles and
turned. You need to keep tapping all the time at the place where the
liquid is going in. You can also tap with a setup where the gel is formed
between two pieces of glass held together with clamps or clips but not
completely sealed, but you probably can't turn or raise the gel much
without it leaking. It helps to have two people, one to keep the gel
pooling at the top and the other to tap. I usually give myself a blister
doing this. Maybe someone else has a better method!

Another thing you can do is de-gas before adding the solidifying agents,
by filtering the liquid and allowing it to stay under the vacuum for an
extra 10 minutes. I'm not sure that really helps, but it used to be part
of our standard procedure.

Sara

---------------------------------------------------
Sara A. Sandstedt, Ph.D.
Department of Epidemiology
School of Public Health
University of Michigan, Ann Arbor

Dr. Eric Rivera-Milla <eric.rivera-milla@uni-konstanz.de
To: Ann Eileen Miller Baker <mouse@lamar.ColoState.EDU
Subject: RE: Other: (1) dnaeasy qiagen expiration; (2) large air bubbles in
acrylamide gels

Ann,
alright.
DNA degradation at -20C (or -80C) is rather estrange to me, but even
likely if you eluted DNA in ddwater instead of the elution buffer. A
bad-quality proteinase could affect DNA storage too. I re-used last
year some opossum samples from 1997 (I think I used a Wizard kit) and
they worker fine for a ~1.4 kbp genomic amplicon. I denature the
aliquot (~30 ul in elution buffer) 5 min at 95C before the PCR and
used fresh primer aliquots. Some times if the primer aliquots are too
old the PCR didn't work.
If there is some degradation you may be able to see it in the agarose gels.
Before new extractions maybe you might consider to do a
re-precipitation of your DNA (EtOh plus salt) and resuspent it in
fresh elution buffer.
I hope that helps you.
Greetings,
Eric

Eric,

When I put this correspondence on the evolution net, it will help
others: none did what you did. Let me try again to be more specific:
and perhaps you don't know because insufficient time has elapsed.
I am PCRing microsatellite loci from genomic mouse dna (trapped mousies).

When you save the AE neat dna in your -20C and wait a year, 5yrs
etc, over long periods (possible degradation occuring) is there a
difference in "PCR-ability" between the new vs 6-yr old dnaeasy kit?

I ask this because some of my AE neat dna now fails to amplify; i.e.,
I might have to go back and re-extract dna (if I can find tissue I
need in the -80C). I made the dna ca. 2002-2003 and periodically (2 or 3X
max I guestimate) defrosted an aliquot of neat AE dna: sometimes
that newly-made aliquot (now 20ng/uL) fails to amplify. I would
re-extract using reagents in my old kits (from ca. 2002-2003).
I try to store dna and tissue @ -80C, though sometimes dna stored
@ -20C; and possibly for a few days @ 9C.

Again thanks,
Ann
======================================

On Tue, 17 Oct 2006, Dr. Eric Rivera-Milla wrote:

 Dear Ann,

 Dr. Rivera-Milla,
 1. thanks

 your welcome.

 2a. when you stored neat AE-dna and then aliquoted for "working stock"
 did your dna work (able to PCR) equally (comparison: dna made from
 new vs old dnaeasy kits?

 yes, that was my surprise. I kept always the first elution at -20 and
 my working solutions is the second elution. My PCRs (RedTad/Sigma)
 worked equally efficiently with genomic (zebrafish caudal fins clips)
 and mitochondrial (opossum liver) DNA, and no differences in
 sequences were detected. I checked 1 ul of the elution in agarose
 gels to see degradation and there was not obvious differences in the
 gels. I used 1-3 ul of the II elution stocks for PCR (20 ul final
 Rx), I checked 5 ul PCR Rx on a gel and used 2 ul for cloning (TOPO
 kit).

 2b. how old (yrs) was the new dnaeasy kit?

 It happened that I looked for the kit in the obvious place without
 finding it, I order a new one and then our TA told me that she saw
 such a kit somewhere in the lab. So, the old one was almost six years
 old, the new one really new. I can not generalize for all kits but
 just try some samples with the old kit and compare the PCR
 amplification using a positive "good" DNA from your collection.

 3. my concern is subtle differences in reagents (new vs old) will
 influencing PCR.

 Your are right, but just try some samples with the old kit you will
 see immediatly in the PCRs.

 Good luck and greetings,
 Eric

 Respectfully,
 Ann
 
 ===== Original Message From "Dr. Eric Rivera-Milla"
 <eric.rivera-milla@uni-konstanz.de =====
 Thanks in advance for your help with two questions; if anyone requests,
 I will post your answers/contact info on the evolution net.
 
 (1) how many yrs after dnaeasy kits expire are the components still
 effective in purifying genomic dna? evidence that genomic dna remains
 stable?
 
 In our lab some kits were "lost" from about 6 years, but they work
  quite fine. We compare them with new ones an surprisingly the yields
 were similar. However, check that some buffers do not form
 precipitate which would affect the extraction yields.
 
 (2) how can I stop getting large air bubbles in gels (6% bis: 
acrylamide=
 formamide-urea-0.5X TBE)?
 
 Try doing a fine milipore filtration, you will see then how the gas
 trapped in the solution fade out (e.g. 0.22 um, GP express Plus
 membrane, MILIPORE, Cat#SCGPT05RE).
 
 
 Ann Eileen Miller Baker
 
 
 
 
 
 Ann Eileen Miller Baker <mouse@lamar.ColoState.EDU

-- ==========
Dr. Eric Rivera-Milla, MSc, PhD
Developmental Neurobiology Group, M-1136
Department of Biology
University of Konstanz
D-78457, Konstanz
Germany

Phones: 0049 7531 88 4088 (office)
0049 7531 88 3121 (lab)
Fax: 0049 7531 88 4863

E-mail: Eric.Rivera-Milla@uni-konstanz.de
ZFIN link:
http://zfin.org/cgi-bin/webdriver?MIval=aa-persview.apg&OID=ZDB-PERS-020304-1
------------------------------

mouse <mouse@lamar.colostate.edu