Probe synthesis for in situ hybThis is essentially according to the method recommended by Boehringer Mannheim. Cut 20 ug of plasmid DNA with appropriate enzyme in 100 ul. Pheno1, phenol/chloroform, chloroform and ethanol precipitate. Dissolve in 15 ul ddH20.
I recommend to add 200 ug/ml Proteinase K after enzyme digestion and incubate 30 min.
1. Mix together2 ul linelized plasmid (1 ug of insert DNA);
2 ul 10 x transcription buffer
2 u1 10 x nucleotide mix
(10 mM each ATP, GTP, CTP, 6.5 mM UTP, 3.5 mM digoxigenin-UTP);
1 ul (20 units) of RNase inhibitor.
11 ul water
2 ul (40 units) T7,, T3 or SP6 RNA polymerase.Incubate the mixture for 2 hour at 37 oC.
10 x Transcription buffer
400 mM Tris-HCl pH 8.0;
60 mM MgC12;
100 mM dithiothreito1;
20mM spermidine;
100 mM NaCl;
RNase inhibitor 1 Unit/ul2. Take 2 ul for elecrophoresis. The probe can be checked on freshly made 1% mini-gel / TAE. The gel box should be washed before use. 2ul rxn mixture should contain 500 ng of RNA.
3. Add 2 ul (40 units) of DNase I and incubate at 37oC for 30 min. to remove the plasmld DNA.
Stop the reaction (optional) by adding 2 ul EDTA (0.2 M pH 8.0) and
Precipitate the RNA with 2.5 u1 4 M LiCl, and 75 ul pre-chilled ethanol for 30 min at ―80 oC.
4. Spin down the pellet and dissolve in 100 u1 of RNAse-free water. Add 900 ul of Hyb solution and store at-20 oC.
We have had success with probes of 0.5-2 kb and signals are stronger if they are not hydrolysed prior to use (requires proteinase K step!). I did not see any improvement by removing the nucleotides e.g. by NucTrap columns.