DIG-Nick Translation Mix Protocol & Troubleshooting

Product No. 11745816910

Protocol

Protocol for chromosome spreads and DNA probes labeled via Nick translation

Reference: Chapter 5 of the Non-radioactive In situ Hybridization Manual (4th ed., p. 76ff).

Detection of human chromosomes in metaphase spreads using DIG or Fluorescein labeled Nick translated Human satellite probes: Reference can be found under the following link.

Troubleshooting

EFFICIENCY / No Signals with FISH

When your FISH shows no signals, first check probe length; if probe length is less than 200-500 bp, check if enough template (minimum 1 μg) was used for labeling. Determine if the protocol used corresponds to the guidelines in the package insert.

Create aliquots (2-3 portions) of the Nick translation Mix solution, because repeated freezing and thawing can degrade their activity.

Roche translation mixes are sensitive to the DNA concentration. Changing the recommended template amount can produce labeled fragments with an increasing size distribution. For this reason, it is important to accurately measure the template concentration, following the guidelines in the package insert of the DIG-Nick Translation Mix. The labeling volume should be 20 μl for each μg of template.

Materials

     
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