CSPD ready-to-use Troubleshooting

Product No. CSPD-RO

Troubleshooting

Low sensitivity

Inefficient probe labeling
Check labeling efficiency of your DIG DNA or RNA labeling by comparison to the labeled control DNA or RNA.

Wrong type of membrane
The quality of the membrane used as support for dot, Southern or northern blotting influences sensitivity and speed of detection. We recommend the Nylon Membrane, positively charged, from Roche Applied Science, specially tested for chemiluminescent detection. Other types of nylon membranes, for example, Biodyne A (Pall) are also suitable but need longer exposure times to X-ray film. Some membranes may cause strong background formation. Nitrocellulose membranes cannot be used with the protocol described.

Inefficient hybridization
Increase the concentration of DIG-labeled probe, but do not exceed 25 ng/ml for DNA probes and 100 ng/ml for RNA probes in the hybridization solution.
Check hybridization and washing conditions.

Low antibody concentration
Make sure, that the recommended dilution of 1:10 000 was used.

Exposure time too short

  • Increase time of exposure to X-ray film
  • The type of film may also influence the observed signal strength.

High background

Inefficient labeling

  • Purify DNA/RNA by phenol/chloroform extraction and/or ethanol precipitation before labeling
  • Make sure that the probe does not contain cross-hybridizing vector sequences.

Wrong type of membrane

  • Although the protocol is optimized for the use of positively charged nylon membranes, some types which are very highly charged can cause background.
  • Lot-to-lot variations in some membranes may also cause problems. When using the recommended Nylon Membrane, positively charged, which is function tested with the DIG system, these problems are avoided.

Concentration of labeled probe too high
Important Note:
It can be necessary to decrease concentration of DIG-labeled DNA or RNA probe. Standard probe concentration for a DNA probe is 25 ng/ml,—for an RNA probe, 100 ng/ml. The critical probe concentration limit (concerning background formation) can be determined by a mock hybridization with increasing probe concentrations using unloaded membrane.

  • Care should be taken not to permit the membranes to dry throughout the whole procedure.

Exposure too long
Shorten exposure time. The signal intensity increases with time.

Materials

     
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