MICROPIPETTE CALIBRATION

Wagner Fontes WAGNERF at BRUNB.BITNET
Fri Sep 25 10:24:51 EST 1992


So, I'm back to the net-pipette

Thanks for everyone who wrote an answer to my problem.
Since I started the discussion, I'm making it's summary.
In the next few lines (almost 600!), there is a SUMMARY of
all the answers I've got until now.

After weighing lots of water, I found out that I have all
kinds of problems.(Pipette-people miscallibrated, leaking and dirty).
Cleaning and replacing rings helps, but it is not everything. The
"miscallibrated people" need recallibration. Curt Ashendel wrote a
procedure for such. Tod Critchlow sent a message about a protocol.
If this protocol contains more information than here, PLEASE consider
this a third request for typing your protocol.


Wagner Fontes                             |   What makes Murphy's
Brazilian Centr of Protein Sequencing     |   Law more evident in
Biochemistry & Protein Chemistry Lab.     |   Biochemistry ?
University of Brasilia - Brazil           |

======================================================================== 18

Date:         Sun, 20 Sep 92 17:27:52 EDT
From: WAGNER FONTES <WAGNERF at BRUNB.BITNET>
Subject:      MICROPIPETE CALIBRATION ?
To: METHODS at genbank.bio.net


HI NET-FOLKS,

   This week I found out that a few micropipetes are out of range
(taking 5 microliters less than they should). Does anybody know how
to calibrate it ? The brand is Gilson, the model is "Pipetman"
(the blue ones, found in almost every lab). Getting specialized service
people here is not an easy task.


   Thanks for any suggestion.

   Wagner Fontes.
======================================================================== 9
Subject: MICROPIPETE CALIBRATION ?
From:    REE700A at MAINE
To:      WAGNERF at BRUNB.BITNET
Date:    Sun, 20 Sep 92 19:41:32 EDT

  lotsa luck...  but replacement parts aren't too expensive (assuming the
plastic seal leaks)

  Jeff Andle   (also available at jandle at wolf.eece.maine.edu)
======================================================================== 34
Date: Sun, 20 Sep 92 19:49 EDT
From: William_D_WARREN at umail.umd.edu (ww40)
Subject: Re: MICROPIPETE CALIBRATION ?
To: WAGNER FONTES <WAGNERF at BRUNB.BITNET>, methods at genbank.bio.net

In my experience it is rare that a pipetman requires recalibration unless it
has been seriously abused.

If I were in your situation I would simply just pull off the tip
ejector and then unscrew the barrel. Inside you will find a stainless steel
plunger assembly (which differ slightly from model to model), a small rubber
O-ring and a small teflon seal (be very careful with these as they are easy to
damage and easy to lose).

Once apart clean the plunger and inspect the O-ring and seal. Also clean out
the barrel, making sure that the surface where the O-ring seals against the
barrel is clean. If the tefon seal or O-ring are damaged then they will
need to be replaced. If your pipettes "drip" then it is likely that the
seals are the problem. If the steel plunger is extremely pitted from corrosion
then you are in trouble. In all likelihood your pipettes are filthy
inside and a good cleaning will probably fix the problem.

In my opinion it is not cost effective to get an inaccurate pipetteman
recalibrated. If it is grossly out then chuck it and buy a new one.

By disassembling and cleaning your pipettes once a month or so
you can prevent damage to the seals and plunger and avoid the need for
costly recalibration or early replacement.  Who knows, even your experiments
might even start consistently working again !!!

Bill Warren
Ctr. Ag. Biotech.
Univ. Of Maryland

======================================================================== 13
Date: Sun, 20 Sep 1992 19:08:50 -0500
From: the End <jgraham at bronze.ucs.indiana.edu>
To: WAGNERF%BRUNB.bitnet at cunyvm.cuny.edu
Subject: Re: MICROPIPETE CALIBRATION ?
Newsgroups: bionet.molbio.methds-reagnts

Wagner,

We weigh out water on the analytical balance as a standard.

Jim
z jgraham bronze  9/20/92
'the End             WAGNERF%BRUNB.bitne  9/20/92*MICROPIPETE CALIBRATION ?
======================================================================== 69
Date: Sun, 20 Sep 1992 21:32:52 EDT
From:         CURT ASHENDEL <ashendel at bchm1.aclcb.purdue.edu>
Reply-To:     ashendel at aclcb.purdue.edu
To:           WAGNERF at BRUNB
Subject: Re: MICROPIPETE CALIBRATION ?


Dear  Wagner Fontes:

	First you must be sure it is out of calibration and not simply a poor
seal, which usually (nerly always) is the cause of this symptom.  To ascertain
this, you must
remove the white shaft by removing the tip ejector (it pulls off), then
unscrewing the blue shaft coupling.  Remove this carefully over a clean bench
as some parts may fall out and get lost if you are not careful.  Unless you hav
a ccache of spare parts (a wise idea, but it sounds like you are a novice with
these devices), such a loss will criple you until an order can be transacted
with Ranin (Gilson distributor in the USA).  It would be helpful if you have
the little booklet that Gilson ships with pipetmen, as it contains an exploded
view of the insides.  At any rate, remove the O-ring/teflon seal combination
from the shaft and then remove the part above it on the shaft.  Then reinstall
the O-ring/seal combo and see how much it clearance it has as you slide it up
and down on the shaft.  If it is snug and cannot shimmy or wiggle and will not
fall off it oriented vertically, then it is OK.  Otherwise, you must place an
order with Ranin (Mack Road, Woburn, MA, if in the US) for the seal for your
size pipetman.  I recommend a sizable number of these (aside from minimum
order requirements) as we wear them out every few months.  DO not repolace the
O-ring, as they never go bad.

	If the seal is OK and it is still pipetting less than the setting, then
it may need recalibrating.  Actually this is a misnomer, since the
"calibration" of the pipetman is fixed by the pitch of the micrometer threads
and can never be "out of calibration".  What can change and be changed is the
limit on the upper end of the travel of the plunger (the one with the button on
it that your thumb touches).  The other thing that can be changed (by mistake)
is the proper remounting of the pair of knurled knobs that flank the rubber
"Friction ring" - but only if it was removed to replace the friction ring
(which crack and fall off after a few years - another worthwhile spare part to
get.)
	Assuming this is not the problem, then to see if recalibration really is
needed, determine the error of the pipettman at each of three or four settings
equally spaced across the range it is capable of measuring.  Recalibration is
called for if the error is constant (or nearly so) in volume.  In your case,
the amount delivered would be expected to be 5 ul low for all settings if it
relly needs recalibration.  As you will see below, it is exceedingly unlikely
that recalibration is needed, because the adjustment cannot become loose.

Recalibration (changing the limit on the upper taravel of the shaft) is done by
using an allen (hex head) wrench to loosen the three or four set screws in the
TOP knurled knob (above the friction ring).  Once loose, a small flat end screw
driver can be used to move the metallic colored slotted collar around the
shaft.  It is threaded and has a very fine pitch, so more than one turn may be
needed.  With the proper tool this wouild be easier to do.  With each
adjustment, try out the pipetman again to see if the correct setting was
obtained.  When obtained, tighten the set screws and check the calibrtion at
each of several volumes.  It should now be correct if this was the problem in
the first place.

	If you need help, call Ranin (if in the US) for tech help over the
phone.  I do not recommend sending it back for service, as they usually tell
you it is too badly worn to repair (not true) and tell you to buy a new one.
For such bad news they charge a small fee.

Good luck. Write if this cannot be interpreted or understood.

	Curt Ashendel
	Dept. Med Chem., Purdue University, W. Lafayette, IN
	Ashendel at aclcb.purdue.edu

======================================================================== 22
Date: Sun, 20 Sep 92 21:52:02 CDT
From: jbh at anat.UMSMED.EDU (James B. Hutchins)
To: WAGNERF at BRUNB.BITNET
Subject: Re: MICROPIPETE CALIBRATION ?
Organization: Dept. of Anatomy, Univ. Mississippi Medical Center


Rainin Pipetmen go back to Rainin (the exact address: Mack Road, Woburn,
MA...don't have zip code here).  They will clean and recalibrate BUT the
price is a significant fraction of replacement cost ($100 and up).

I have seen ads for others who do this but do not remember their names
now.  Midwest Scientific in St Louis, MO comes to mind but I'm not sure.
Perhaps others can give more specific info (w/ experiences) in this
regard.

Jim

--
Jim Hutchins                    []     E-Mail: jbh at anat.umsmed.edu
Dept of Anatomy                 []
Univ Mississippi Med Ctr        []
======================================================================== 13
To: WAGNERF at BRUNB.BITNET
From: "Ed Rybicki"  <ED at micro.uct.ac.za>
Date:     21 Sep 92 09:55:49 SAST-2
Subject:  Re: MICROPIPETE CALIBRATION ?
Reply-To: ed at micro.uct.ac.za


You can get a self-calibration kit from Gilson with no hassle - we
standardised on them long ime ago, and got a couple of kits, and do all
our own pipettes (use them for student practicals, the works).  You think
you got problems getting service staff...we're 12 hours flying time from
the nearest Gilson service centre!

======================================================================== 13
Date:         Mon, 21 Sep 92 10:46:43 +0200
From:         "D.D.Bilanovic" <IBT329 at DJUKFA11>
Organization: Forschungszentrum Juelich GmbH
Subject:      Re: MICROPIPETE CALIBRATION ?
To:           WagnerF <WAGNERF at BRUNB>
In-Reply-To:  Your message of Sun, 20 Sep 92 21:42:38 UT

1. Adjust piston position
2. Calibrate with dest.H2O (i.e. analytical balance)
3. Go to No.1.
Dragoljub
z IBT329 DJUKFA11  9/21/92
'D.D.Bilanovic       WagnerF              9/21/92*MICROPIPETE CALIBRATION ?
======================================================================== 31
To: methods-and-reagents at genbank.bio.net
From: binnie at aclcb.purdue.edu (Al Binnie)
Subject: Re: MICROPIPETE CALIBRATION ?
Date: 21 Sep 92 14:35:47 GMT
Sender: news at mentor.cc.purdue.edu (USENET News)
Reply-To: binnie at aclcb.purdue.edu

William_D_WARREN writes:

>In my experience it is rare that a pipetman requires recalibration unless it
>has been seriously abused.

In my opinion, this is an extreme inaccuracy that could cause much frustration.
In my experience, pipetmen slip out of calibration constantly with normal use.
If you don't check the accuracy (by the aforementioned weighing of water
delivered on a good balance, for instance) quite frequently (at least once a
month) you are asking for serious trouble.
>
>By disassembling and cleaning your pipettes once a month or so
>you can prevent damage to the seals and plunger and avoid the need for
>costly recalibration or early replacement.  Who knows, even your experiments
>might even start consistently working again !!!

Yes!  A good cleaning will often help restore accuracy and should be done
regularly, whether they "need it" or not.
                       al binnie......
                                      Purdue University
                                      Department of Biochemistry
                                      Internet: binnie at aclcb.purdue.edu
                                      317-494-1658 (W)

======================================================================== 40
Date: 21 Sep 92 11:51:19 U
From: Mark Guiltinan <mark_guiltinan at agcs.psu.edu>
Subject: RE: MICROPIPETE CALIBRATION ?
To: WAGNERF at BRUNB.BITNET

Call the company, they are very helpful.

Mark Guiltinan
Penn State Biotechnology Institute
mjg at psupen.psu.edu
======================================================================== 51
To: methods-and-reagents at genbank.bio.net
From: tcritchl at sdcc3.ucsd.edu (Tod Critchlow)
Subject: Re: MICROPIPETE CALIBRATION ?
Date: 21 Sep 92 16:50:49 GMT


Somebody else suggested throwing out an out of calibration pipetman.

DON'T DO THAT.  IT IS POSSIBLE TO RECALIBRATE A PIPETMAN.

Bill Warren had a good discription of cleaning the plunger, barrel
and o-rings.  I have a copy of a protocol for the whole
process, including instructions on how to calibrate the things.

I believe Gilson also sells a tool for adjusting the calibration
on their newer models.  For the older models, an allen wrench and
a screw driver are all that is needed.

I will try to transcibe a copy of the protocol here,
but if anyone else knows of an electronic copy of a
similar protocol, it will save me much typing.


--
_____________________________________________________________________
Tod Critchlow(tcritchl at ucsd.edu)      |  What the Hell is a
Univ of Calif, San Diego              |  Signature anyways??
Scripps Institution of Oceanography   |

======================================================================== 33
Date: Mon, 21 Sep 1992 13:17:00 EDT
From: "Bill Melchior, NCTR/FDA" <wmelchior at NTBTOX.NCTR.FDA.GOV>
Reply-To: wmelchior at NTDOC.NCTR.FDA.GOV
To: methods at genbank.bio.net
Subject: Re: MICROPIPETE CALIBRATION ?

>Somebody else suggested throwing out an out of calibration pipetman.

 ... DELETED

Tod-

If you can bring yourself to do the typing, it would be much appreciated.
I have recalibrated early Pipetmen (Pipetmans -- what IS the plural?)
according to instructions that came with them, but they stopped including
those instructions with the instruments MANY years ago, and I've wished that
I'd copied them when I was in the lab that had them.

Since Rainen has a service department, I assume the reason for not including
those instructions is monetary.  And I suspect that this is the reason
Spectrum stopped sending clear instructions for cleaning Spectropor
dialysis tubing.  Their instructions used to give common lab chemicals, but
now call for Solution 1 and Solution 2 (available, of course, from Spectrum).

________________________________________________________________________________
The opinions stated are mine, not those of NCTR or its sponsoring organizations.

Bill Melchior                                ||     OMNISCIENCE
National Center for Toxicological Research   ||    Knowing what
Jefferson, AR  72079                         ||    thou knowest not
(501) 543-7206                               ||    is in a sense
                                             ||    omniscience.
WMELCHIOR at NTDOC.NCTR.FDA.GOV                 ||       from Grooks, Piet Hein
======================================================================== 8
To: methods at genbank.bio.net
Subject: Re: MICROPIPETE CALIBRATION ?
Date: Mon, 21 Sep 92 15:11:51 -0400
From: lev at dfciaids.dfci.harvard.edu

Unfortunatuly, the new generation of Pipetmens by Rainin (in contrast
to the old French? ones) could not be recalibrated without special tools.
                                                                    Lev
======================================================================== 23
To: methods-and-reagents at genbank.bio.net
From: bchs1b at Elroy.UH.EDU (Michael Benedik)
Subject: Re: MICROPIPETE CALIBRATION ?
Date: 21 Sep 92 19:13:02 GMT
Sender: usenet at menudo.uh.edu (USENET News System)
Reply-To: bchs1b at Elroy.UH.EDU

In article <00960F38.956974E0.456 at NTBTOX.NCTR.FDA.GOV>, wmelchior at NTBTOX.NCTR.FD
A.GOV ("Bill Melchior, NCTR/FDA") writes:
>>Somebody else suggested throwing out an out of calibration pipetman.
>
   ... DELETED

I second the request to type in the instructions. PLEASE.
To answer Bill's question, the plural of Pipetman is obviously
Pipetpeople. (I also prefer Pipetperson for the singular).

----------------------------------------------------------------------
 Michael Benedik                                INTERNET: Benedik at uh.edu
 Dept. of Biochemical & Biophysical Sciences
 University of Houston                          BITNET: Benedik at uhou
-----------------------------------------------------------------------

======================================================================== 25
To: methods-and-reagents at genbank.bio.net
From: wetsel_r at wums.wustl.edu
Subject: RE: MICROPIPETE CALIBRATION ?
Date: 22 Sep 92 00:24:40 GMT

Yea... it depends upon the pipetter... if it's one of the older ones you can
do it.  How do you tell?  If the pipetter has hex nuts on the adjustment dial
then you have an older one that can be calibrated if necessary.  The newer ones
take a "forked" tool that fits into the  two holes on the top of the
adjustment dial - that tool is very expensive from Gilson - that is, they
would rather not sell it to you!  We were quoted a price for that tool where
we could have purchased another pipetter!  Fortunately, most of ours have not
needed calibration if you take a few simple steps.  1) every year, replace
ALL "O" rings, 2) disassemble and clean all internal parts, and 3) we lube it
with a small, sparing, amount of silicon vacuum grease.  Gilson, for some
reason, doesn't recommend step 3 but we've never had any problems with it.
also, ANY time you notice anything getting sucked up into your P-1000 like SDS,
or GTC, disassemble and clean it on the spot.  then, just so you know what
 the delivery is, do as a previous poster suggested - pipet 50% and 100% of
rated volume of DDH2O onto an analytical scale and determine the percent
error.  We've found that after cleaning, most Gilsons are within a 1-2%
tolerance... if not, we have to send it out...

Hope that helps...

======================================================================== 33
To: methods-and-reagents at genbank.bio.net
From: anita at sandman.caltech.edu (Anita Gould)
Subject: Re: MICROPIPETE CALIBRATION ?
Date: 22 Sep 92 06:41:47 GMT

In article <22SEP92.00244003 at wums.wustl.edu> wetsel_r at wums.wustl.edu
writes:

 > YEA... IT DEPENDS UPON THE PIPETTER... IF IT'S ONE OF THE OLDER
   ... DELETED

To calibrate the newer kind, I don't think you need to buy their
exorbitantly-priced special tool.  We use a tool with plier-like
handles and blunt pins on the end of the jaws that my labmate says he
got at Fedco (local discount dept store).  Gilson did not invent the
concept of a wheel turned in this fashion, so there ought to be
generic tools available.  And if you don't want to spring for one
of those, you might be able to use a long-nosed pliers end-on.
Anyway, to calibrate the thing, all you need to do is to turn the
inner wheel at the top (the one with the six equidistant holes in it)
relative to the outer one (the fingerwheel used to dial the volume).
As far as I can tell, the two are just friction-fitted together (if
anyone knows differently, please enlighten us!), so all you need is
something that will fit into 2 diametrically opposed holes in the
inner wheel and exert enough torque to turn it.  It's easiest to get
at if you first dial the Pipetman to maximum volume, and the plunger
cap just pulls straight off (yes, reversibly!) to facilitate access.

Good luck!
--

                                   -Anita Gould
                                    anita at cco.caltech.edu
======================================================================== 25
Date: 22 Sep 92 10:41:23 U
From: "Mark Guiltinan" <mark_guiltinan at agcs.psu.edu>
Subject: RE: MICROPIPETE CALIBRATION ?
To: methods at genbank.bio.net, lev at dfciaids.dfci.harvard.edu

Actually, you can use the metal wire from a disassembled small or medium sized
binder clip (same type as used for sequencing gels but smaller).  You can use
the end to insert into the hole of the inner ring on top of the adjustable
dial.  Hold the inner ring still using the clip while turning the outer dial.
This will change the calibration set.  What do you think, maybe we should
publish this in Biotechniques (ha ha)?

Mark Guiltinan
Penn State Biotechnology Institute
mjg at psupen.psu.edu
_______________________________________________________________________________
From: lev at dfciaids.dfci.harvard.edu on Mon, Sep 21, 1992 3:38 PM
Subject: RE: MICROPIPETE CALIBRATION ?
To: methods at genbank.bio.net

Unfortunatuly, the new generation of Pipetmens by Rainin (in contrast
to the old French? ones) could not be recalibrated without special tools.
                                                                    Lev


======================================================================== 23
Date: 22 Sep 1992 10:47:17 -0500 (EST)
From: "DAVID F. BISHOP" <BISHOP at msvax.mssm.edu>
Subject: Re: MICROPIPETE CALIBRATION
To: methods-and-reagents at genbank.bio.net

For the last 10 years, we have never sent out Pipetmen for recalibration.
Sufficient accuracy has been maintained by replacing the teflon and rubber
seals (always at the same time) every 6-12 months.  If the seal isn't tight
enough to keep the plunger from slipping out by gravity, it's time to
replace them.  We never use silicon grease, but carefully clean the stainless
steel plungers and nose pieces.  Ocassionaly a nosepiece must be replaced
if it developes a hairline split (how many times do you hear a $200 Pipetman
fall to the floor?). The only time a pipetter is sent back for repair is
when the stainless steel plunger is pitted or badly scratched. (Cost ca 50%
of a new one).  It is good to put these Pipetmen on a regular maintenance
schedule for cleaning and seal replacement.  Most people in the lab seem to
go too long before checking.

David Bishop                             | EMail:Internet:Bishop at msvax.mssm.edu
Div. of Medical and Molecular Genetics   |         Bitnet:Bishop at msrcvax
Mount Sinai School of Medicine, Box 1203 | Phone: (212) 241-6946
Fifth Avenue and 100th Street            | FAX:   (212) 360-1809
New York, NY 10029                       | GEnie: D.Bishop
======================================================================== 28
To: methods-and-reagents at genbank.bio.net
From: afc at gnv.ifas.ufl.edu
Subject: RE: MICROPIPETTE CLEANING
Date: 23 Sep 92 18:11:02 GMT

In regard to the thread on micropipette calibaration:

Several people have mentioned that they do not bother recalibrating;
they just clean carefully several times a year.  I have a horror story
that has induced our lab into careful, frequent cleaning...

For the past year, we have been having trouble with plasmid contamination
on our genomic southern blots.  Sometimes one lane is affected, sometimes
an entire gel.  I screamed and pleaded for people to be careful,threw out
all of the solutions in the lab several times, but the problem persisted.

Finally I had the bright idea that our pipettepeople might be infested
with plasmid DNA.  After washing, the problem disappeared.  What had
happened was that the person making our plasmid preps was using the P1000
to resuspend pellets, and massively contaminating the pipette barrel.
People doing genomic DNA preps were occassionally getting a little solution
into the barrel and randomly contaminating their preps.  Now we wash
them regularly, and keep separate pipettors for plasmid and genomic DNA
(we already had a separate set for PCR).

Andrew Cockburn
USDA

======================================================================== 38
Date: Thu, 24 Sep 1992 09:18:36 +1000
To: methods-and-reagents at genbank.bio.net
From: Klaus.Matthaei at anu.edu.au
Subject: RE: MICROPIPETTE CLEANING

>In regard to the thread on micropipette calibaration:
 ... DELETED

G'Day

Ever wondered what those so called 'deletions' are when you subclone a
fragment and get vector only even if properly phosphatased?  Guaranteed
Gilson contamination.
We routinely decontaminate our Pipetpeople (yes we have been using
non-sexist language for some time also) to overcome this problem.  We used
to depurinate the barrels with HCl followed by NaOH (0.1N and 0.4N resp.)
but now treat with sodium perchlorate (^5% for 5 min, see ref. that escapes
me at the moment in a Biotechniques issue suggested for PCR
decontamination) and have reduced, if not eliminated the problem (sloppy
people still exist).

Hope this helps.

Cheers, Klaus

PS:  5X loading dye is also a great source of spurious bands in southerns
etc.from the same source and multi users.

--------------------------------------------------------------------------
Klaus Matthaei
Gene Targeting
The John Curtin School of Medical Research
The Australian National University
E-mail: Klaus.Matthaei at anu.edu.au

"If all else fails.  Read the instructions".
--------------------------------------------------------------------------

======================================================================== 27
To: methods-and-reagents at genbank.bio.net
From: pnh at fcsparc6.ncifcrf.gov (Paul N Hengen)
Subject: Re: MICROPIPETE CALIBRATION ?
Date: 24 Sep 92 19:40:47 GMT

In article <22SEP92.00244003 at wums.wustl.edu> wetsel_r at wums.wustl.edu writes:

: ... do as a previous poster suggested - pipet 50% and 100% of
:rated volume of DDH2O onto an analytical scale and determine the percent
:error.  We've found that after cleaning, most Gilsons are within a 1-2%
:tolerance... if not, we have to send it out...

This might work just fine for a P1000, but the P20 pipetman can be off
by a significant amount depending on where you've done the calibration,
ie. at the 1 - 5 ul mark or the 15 - 20 ul mark. I suggest that you
calibrate a P20 near the level of normal use. For example, I nearly
always use my P20 for measuring 1 - 5 ul and use a P200 for 20 - 50 ul.
For the P20, I calibrate it at 3 ul. This is important if you do enzyme
reactions requiring say 1 ul of 10X buffer. If you calibrate at 20 ul,
you'll see that it'll be off at the lower volumes.

Paul N. Hengen
National Cancer Institute
Frederick Cancer Research and Development Center
Frederick, Maryland 21702-1201 USA



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