Make DNA templates via PCR

Day 1

It's preferable to start with constructs that contain RNA polymerase start sites (T3, T7, or SP6) which allow use of primers outside the start-point. This gives the enzyme some docking space, improving transcription efficiency. If this is not possible, use extended primers with RNA-polymerase start sites in their 5' tails.

Purify PCR products with QiaQuick PCR purification kit and resuspend in nuclease free water.Make DNP-NTP mixture (if needed)

Day 1

In vitro transcription reaction

Day 1

Heat 10X Buffer to dissolve salts before using.

Incubate at 37oC for 2-4 hours.

Eliminate template

Day 1

Add 1 μl DNAseI (RNAse free) to each reaction.

Incubate for 15 min at 37oC. This is a time sensitive step.

Precipitate probes

Day 1 (immediately after eliminating the template)

Bring reaction to 50μl with RNAse free water.

Add 1/10th volume 3M NaOAc pH5.2 (5μl)

Add 2.5 volumes of 100% EtOH (125μl)

Store at -20oC for at least 2 hours.

Spin 20 min at 4oC at 13200 rpm.

Remove supernatant.

Pellet for DIG/biotin reactions will be white, DNP reactions will be yellow and FITC reactions will be orange.

Add 250-400μl 70% EtOH and spin 20 min at 4oC at 13200 rpm.

Remove supernatant and repeat wash with 100% EtOH.

For a 20μl reaction with a visible pellet, it is usually sufficient to dissolve the pellet directly in 100μl 50% formamide.

If necessary: resuspend probes in 50 μl of RNAse free water. Take 5 μl for quantitation. Then add 45 μl

100% formamide and store at -20oC.

For 10μl reaction : with visible pellet, it is sufficient to dissolve them directly in 50μl 50% formamide in RNAse free water.

Properly dissolved probes that have sufficiently high concentrations typically foam a lot. It will take a couple of minutes to dissolve a probe.

Making RNA probes for in situ hybridization page 2of 2