in vivo link kit from topogen: modifications

ABSTRACT:

We have modified the In Vivo Link Kit to make it easier. Before we formally incorporate these modifications, we are posting them on the Web to assist our customers in transitioning to the new technology. While the new and improved method still relies on CsCl fractionation, we now use a single cushion of CsCl through which the lysate is pelleted. The DNA is recoverable from a single pellet, resuspended in buffer and the concentration measured. Several different concentrations of DNA are then blotted onto Nitrocellulose followed by probing with TopoGEN antibodies.

Modified ICT Bioassay: pelleting protein-DNA complexes through cesium chloride*

Lysates- 2 ml volume. Tested with following cells:

Hela cells: 100 mm dish/ gradient
MCF-7: 2- 100 mm dishes/ gradient

*Note: In applying this new method, be sure to perform a test run with +/- drug to verify that it is working in your hands in your lab.

Gradients:

Make 1.5 g/cc density cesium from your stock 1.8g/cc (refractive index = 1.414).

With a 1cc syringe and 18 gauge needle, add 1.5 ml cesium into a 3.5 ml sealtop ultracentrifuge tube.

Layer your 2 ml lysate on top until the base of the neck using either a pasteur pipette or syringe with 18 gauge needle. Save any remaining lysate to balance.

Seal with heat sealer or use metal caps on tops to melt with hot sealer. Hold down until cool with metal holder. Squeeze gently to check for leaks.

Be aware of which side of the tube is facing out on the rotor (where your pellet will form; in fixed angle rotor, DNA will be on the side of tube).

Centrifuge 5 hours at 90,000 rpm (438,000 x g for r-max in quick seal tube).

Set for maximum acceleration but no deceleration - set at ``0". Takes about 15-20 minutes to stop.

To Retrieve Sample:

Pellet may not be visible through the tube until the medium is removed- pellet will be clear.

Cut the top of the tube off.

Use a pasteur pipette or syringe with 18 gauge needle to remove all the liquid from the tube.

Cut the tube in half and invert to drain.

Rinse pellets with 100% ETOH (rt) by swirling ethanol over the pellet and pouring off- (keep your eye on the pellet and make sure it is not moving).

Drain and invert, allow to dry briefly, until you have finished all your samples.

Resuspend the DNA in a suitable amount of water or TE and transfer to an eppendorf tube. Usually 200 ul yields 0.5-1 ug/ul for 1 Hela plate or 2 MCF-7 plates.

Heat the DNA at 65 C for 5-15 minutes alternating with vortexing for several rounds to dissolve the DNA in solution or preferably place in the cold room overnight with gentle agitation.

Read the absorbance at 260 nm. For DNA, 1 OD unit corresponds to 50 ug/ml. Usually, you will need to dilute an aliquot anywhere from 1:100 to 1:30 to get a suitable reading in the linear range.

After the DNA concentration is determined, slot blot 3-4 different DNA concentrations on Hybond Nitrocellulose. Typically we blot:
0.5, 1, 5 ug of DNA.

Process and probe as per protocol for Western Slot blots.

We offer custom screening on a contract basis. Let us test your compounds for topo targeting activity.