Chrombios Molecular Cytogenetics

This is a typical protocol for the preparation of chromosomes from adherant cell cultures such as fibroblasts and mouse ES cells.

Materials:

Colcemid: stock solution 10µg/ml (Gibco, 15212-012)

Hypotonic solution: 75mM KCl. (0.56%) in water

Fixative: mix methanol and acetic acid 3:1 before use. Fixative can be stored at -20 C for few days.

Procedure:

Most importantly, cells should grow exponentially. In case they are already in stationary phase no mitotic divisions and thus no cytogenetic analysis can be obtained.

Add colcemid (0.2 µg/ml culture medium, i.e. 20 µl/ml medium), incubate for 1h.

Trypsinize cells as usual, transfer into a 15ml tube and spin down at 200xg (about 1100 rpm in most lab centrifuges) for 10 min. Remove supernant except for about 0.5 ml.

Resuspend the pellet by gently flicking the tube. Add 10 ml hypotonic solution dropwise while gently flicking the tube. Leave for 10 min at room temperature, spin down cells at 1100 rpm for 10 minutes.

Remove supernant except for about 0.5 ml. Resuspend the pellet by gently flicking the tube. It is very important that cells are completely resuspended. However, do not use a pipette! Add 2-3 ml fixative dropwise while gently flicking the tube. Fill up the tube with fixative (15ml) and leave it in a freezer for 30 min.

Spin down at 1500 rpm for 10 min. Remove supernant except for about 0.5 ml. Resuspend the pellet by flicking the tube. Add 2-3 ml fixative dropwise while gently flicking the tube. Fill up the tube again with fixative.

At this point cells are ready for transport.

Transport:

You may spin down the cells again and resuspend them in about 1ml fixative for transportation in a microtube with a screw cap with a rubber ring. Take care that the tube is securely sealed. Any leaking of the fixative may remove the labeling of the tube. You may wrap the tubes separately in small plastic bags and label the bags at the outside. Alternatively, you may place the microtube into a 15ml plastic tube.Place another label on the outside of the tube.

When sending by express mail cells do not need dry ice. A cooling element is sufficient.

For further information and pricing call at +49 8035 984928
FAX +49 8035 984930
Email: chrombios@chrombios.com

	.......Protocol for Chromosome Preparation


			This is a typical protocol for the preparation of chromosomes from adherant cell cultures such as fibroblasts and mouse ES cells. Materials: Colcemid: stock solution 10µg/ml (Gibco, 15212-012) Hypotonic solution: 75mM KCl. (0.56%) in water Fixative: mix methanol and acetic acid 3:1 before use. Fixative can be stored at -20 C for few days. Procedure: Most importantly, cells should grow exponentially. In case they are already in stationary phase no mitotic divisions and thus no cytogenetic analysis can be obtained. Add colcemid (0.2 µg/ml culture medium, i.e. 20 µl/ml medium), incubate for 1h. Trypsinize cells as usual, transfer into a 15ml tube and spin down at 200xg (about 1100 rpm in most lab centrifuges) for 10 min. Remove supernant except for about 0.5 ml. Resuspend the pellet by gently flicking the tube. Add 10 ml hypotonic solution dropwise while gently flicking the tube. Leave for 10 min at room temperature, spin down cells at 1100 rpm for 10 minutes. Remove supernant except for about 0.5 ml. Resuspend the pellet by gently flicking the tube. It is very important that cells are completely resuspended. However, do not use a pipette! Add 2-3 ml fixative dropwise while gently flicking the tube. Fill up the tube with fixative (15ml) and leave it in a freezer for 30 min. Spin down at 1500 rpm for 10 min. Remove supernant except for about 0.5 ml. Resuspend the pellet by flicking the tube. Add 2-3 ml fixative dropwise while gently flicking the tube. Fill up the tube again with fixative. At this point cells are ready for transport. Transport: You may spin down the cells again and resuspend them in about 1ml fixative for transportation in a microtube with a screw cap with a rubber ring. Take care that the tube is securely sealed. Any leaking of the fixative may remove the labeling of the tube. You may wrap the tubes separately in small plastic bags and label the bags at the outside. Alternatively, you may place the microtube into a 15ml plastic tube.Place another label on the outside of the tube. When sending by express mail cells do not need dry ice. A cooling element is sufficient. For further information and pricing call at +49 8035 984928 FAX +49 8035 984930 Email: chrombios@chrombios.com .

	Chrombios GmbH • Mühlenstr.1 • D-83064 Raubling • Tel.: +49 (0) 8035/984928 • Fax: +49 (0) 8035/984930 • chrombios@chrombios.com• www.chrombios.com