cloning cylinders

[Bernard Murray, PhD]

In article <l03130303b4e4d4016ddd@[129.127.90.187]>,
adob...@medicine.adelaide.edu.au (alex dobrovic) wrote:

> Can someone tell me who distributes glass cloning cylinder for subcloning
> cell lines.

In the short term in dishes I have used sterile 1 ml pipette tips
(big end downwards).

> Also what type of grease can be used besides Corning high vacuum.

I've just taken "Vaseline" petroleum jelly and autoclaved some
in a shallow glass dish.

Bernard

--
Bernard P. Murray, PhD
bpmurray at cgl . ucsf . edu
Department of Cellular & Molecular Pharmacology, UCSF

[alex dobrovic]

Can someone tell me who distributes glass cloning cylinder for subcloning
cell lines.

Also what type of grease can be used besides Corning high vacuum.

Regards

Alex

adob...@medicine.adelaide.edu.au

---

[Jens Tornoe]

When I first learned to isolate clones, I was taught to use the glass
cloning cylinders. I found them difficult to handle and I have later
abandoned them. Now I simply remove the cells with a p100 pipette from a
dish / flask where I have aspirated most of the medium. It is fast and
convenient, and the cells appear to take no damage. With a little pratice,
one can successfully lift almost all cells from each colony using this
method.

Jens Tornře
NsGene, Denmark

alex dobrovic <adob...@medicine.adelaide.edu.au> wrote in message
news:l03130303b4e4d4016ddd@[129.127.90.187]...

[Kiley R. Prilliman]

...I do the same thing, works like a charm.

Kiley R. Prilliman, Ph.D.
Postdoctoral Research Fellow
Division of Immune Regulation
La Jolla Institute for Allergy & Immunology

ki...@liai.org

[Jan-Henner Wurmbach]

Jens Tornoe <je...@tornoe.net> schrieb im Beitrag
<38Xv4.766$i3.3...@news0.mobilixnet.dk>...

> When I first learned to isolate clones, I was taught to use the glass
> cloning cylinders. I found them difficult to handle and I have later
> abandoned them. Now I simply remove the cells with a p100 pipette from a
> dish / flask where I have aspirated most of the medium. It is fast and
> convenient, and the cells appear to take no damage. With a little
pratice,
> one can successfully lift almost all cells from each colony using this
> method.
>
> Jens Tornře
> NsGene, Denmark

Can you please write that down in a longer version?
It seems to be the solution to my biggest Problem.
Thank you

Jan-Henner Wurmbach

[Jens Tornoe]

It is a little difficult to write a longer version, since there
really isn't more to the method than I have already
described...

Just let your colonies form in your selection medium as you
would do when using cylinders. When they are ready to be picked,
mark the position of the colonies you wish to isolate, remove
most of the selection medium and find your favorite pipette (100-
200 ul). For each colony, place a pipette tip on top of the
colony and gently scrape off the colony with the tip while
sucking up media. Try to cover all of the colony surface in one
go. Transfer the cells to a suitable culture dish, ét viola: you
have isolated your first colony! Continue ad libitum.

Hope this version is long enough for you. ;-)

Best of luck

Jens

------------
Jens Tornÿfffff8

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[Cornelius Krasel]

Jens Tornoe <je...@tornoe.net> wrote:
> When I first learned to isolate clones, I was taught to use the glass
> cloning cylinders. I found them difficult to handle and I have later
> abandoned them. Now I simply remove the cells with a p100 pipette from a
> dish / flask where I have aspirated most of the medium.

This works very well if the cells are only loosely attached (e.g. with
HEK293 cells) but I haven't managed to get it to work with cells which
are strongly attached to the dish. In this case, we try to subclone by
dilution. (What are glass cloning cylinders?)

--Cornelius.

--
/* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */
/* D-97078 Wuerzburg, Germany email: pha...@rzbox.uni-wuerzburg.de SP4 */
/* "Science is the game we play with God to find out what His rules are." */

[Jens Tornoe]

You are right. I would not use the pipetting method for picking
colonies from cells like MDCK or the like. For those, I have
used the cloning cylinders. They are small glass cylinders that
you make fit around a colony with grease so you can trypsinize
the colonies individually, so to speak.

Whether this is a better and more convenient method than
dilution, I will let you to decide. The cylinders work, but they
are a pain to work with, IMHO.

Jens

------------
Jens Tornoe
NsGene
Ballerup, Denmark

[Ashok Aiyar]

In article <glj0a8...@wpxx02.toxi.uni-wuerzburg.de>, Cornelius Krasel
<kra...@wpxx02.toxi.uni-wuerzburg.de> wrote:

> Jens Tornoe <je...@tornoe.net> wrote:
> > When I first learned to isolate clones, I was taught to use the glass
> > cloning cylinders. I found them difficult to handle and I have later
> > abandoned them. Now I simply remove the cells with a p100 pipette from a
> > dish / flask where I have aspirated most of the medium.
>
> This works very well if the cells are only loosely attached (e.g. with
> HEK293 cells) but I haven't managed to get it to work with cells which
> are strongly attached to the dish. In this case, we try to subclone by
> dilution. (What are glass cloning cylinders?)

Here's an alternative method that works well for cells that adhere
strongly to dishes.

a) Cut Whatman 3m filter paper into tiny squares (0.25 sq centimeters).
Alternatively just punch out circles with a paper punch.
b) Autoclave these filters in a petri-dish.
c) Place a few filters on a clean petri-dish lid, and put a few drops
of trypsin on them. Setup a petri-dish with 10 mls of 70% ethanol,
and another with 10 mls of PBS.
d) Mark the colonies you wish to clone by drawing circles around them.
e) Aspirate the medium off the plate that contains the colonies.
f) Dip the ends of a clean pair of forceps in the 70% ethanol, and then
in PBS. Shake off the excess PBS.
g) Now pick up a piece of trypsin-soaked filter paper and overlay it on
a colony.
h) Repeat for the other colonies you wish to clone on the same plate.
i) After a couple minutes pick up the filter paper with the forceps
and drop it with a little agitation into a well of a multi-well
plate that already has the appropriate medium in it.
j) Change the medium after two days, and remove the filter paper at
that time (it will stick to the end of the pasteur pipette if the
vacuum is on).

I learned this method from my post-doctoral mentor, Bill Sugden, and
have used it several times. It is much easier than using cloning
cylinders, and doesn't require you to clone by limiting dilution.

Regards,
Ashok

--
Ashok Aiyar
Assistant Professor Email: a-a...@nwu.edu
Department of Microbiology-Immunology Tel: (312) 503 2524
Northwestern University, Chicago, IL 60611 Fax: (312) 503 1339

[Klaus Lehnert]

Well, I always use the method described by Jan-Henner to pick my MDCK, CHO
and other well sticking transformants. Just make sure they don't dry out.
For that reason, I'll try to have less than 50 colonies per 9cm-dish,

Klaus Lehnert

Jan-Henner Wurmbach <wurm...@uke.uni-hamburg.de> wrote in message
news:01bf8777$aba5efe0$c7606486@default...

>
>
> Jens Tornoe <je...@tornoe.net> schrieb im Beitrag
> <38Xv4.766$i3.3...@news0.mobilixnet.dk>...

> > When I first learned to isolate clones, I was taught to use the glass
> > cloning cylinders. I found them difficult to handle and I have later
> > abandoned them. Now I simply remove the cells with a p100 pipette from a