Thermo Scientific Shake 'n Stack 6244, Operating And Maintenance Manual

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© Thermo
Scientific
, May 2003. Issue 7 20
CHAPTER 5
HYBRIDISATION GUIDE
NON-RADIOACTIVE SYSTEM
The use of non-radioactive labelling techniques is becoming increasingly important for
nucleic acid Hybridisation. There are currently a number of systems available. Most are
based on the labelling of probes with either biotin or the hapten Digoxygenin (DIG).
Biotinylated probes are hybridised and then linked to an appropriate enzyme such as
alkaline phosphatase. Visualisation is achieved by reaction of the enzyme with a
chromogenic substrate.
Similarly, DIG labelled probes are detected by high affinity Fab fragments coupled to
alkaline phosphatase. Visualisation is achieved using chromogenic substrates such as
NBT and BCIP, which produces a dark blue colour.
Although both of these systems offer advantages over radio-labelled probes (no safety
risk or stability problems), they have been recognised as less sensitive.
Recently the sensitivity of detection has been increased significantly (down to
femtogram levels) by using a chemiluminescent reaction. The Böehringer DIG system
with the same alkaline phosphatase conjugate will generate light with the
chemiluminescent substrate AMPPD. The resultant signals produced can be detected
using standard X-ray film or a phosphoimager in as little as 30 minutes. Light continues
to be given off for up to 48 hours for longer developments.
The DIG System in Thermo Hybridisation Ovens
The small probe volumes, even mixing, accurate temperature control and ease of
handling associated with Thermo Oven systems are equally suited to the success of
non-radioactive Hybridisation.
The protocol detailed below has been developed in conjunction with the Böehringer DIG
system. The prehybridisation, Hybridisation and washing stages of the protocol using the
DIG labelled probe are exactly as for radio-labelled probes. The subsequent wash
stages associated with detection and visualisation of the probe is often performed in
sandwich boxes, but will work equally well in bottles.
This protocol has been optimised for DNA RFLP analysis. It may require minor alteration
to be used for other applications.
The following protocol applies to a 20cm x 20cm membrane: -