Method prepared by Grace Panganiban: firstname.lastname@example.org
RNase-free solutions needed:
10X transcription buffer* 10X dig U NTP mix*
0.4 M Tris pH 7.5 10 mM ATP
60 mM MgCl2 10 mM GTP
100 mM NaCl 10 mM CTP
20 mM spermidine-HCl 6 mM UTP
4 mM dig-UTP
*These solutions can be purchased ready made from Boehringer Mannheim
2X carbonate buffer Stop solution
120 mM Na2CO3 0.2 M NaAc
80 mM NaHCO3 pH to 6.0 with HOAc
pH to 10.2
Aliquot and store at -20oC
50 mM DTT 4M LiCl
DEPC-treated deionized water EtOH
20 mgs/ml glycogen
T3, T7, or SP6 RNA polymerase
RNase-free DNase I
1) Linearize 5-10 ugs of template DNA completely with an enzyme which cuts at the 5' end of the insert in pBS and which does not leave a 3' overhang.
2) Add proteinase K to 200 ug/ml and incubate at 37oC for 30 min.
3) Add 1/4 volume of 10 M NH4Ac.
4) Extract with an equal volume of phenol:chloroform:isoamyl alcohol.
From this point on, handle the DNA with RNase-free tips and use RNase-free reagents.
5) EtOH precipitate with 2 volumes of EtOH. Spin for 10 min. Rinse pellet with 70% EtOH. Air dry.
6) Resuspend DNA in 20 ul diw. Quantitate on a slide gel or on a spec.
1 ug DNA (in less than 4 ul diw)
2 ul 10X transcription buffer
2 ul 10X dig mix
2 ul 50 mM DTT
2 ul RNasin (optional)
diw to 18 ul
2 ul T3, T7, or SP6 polymerase
Incubate at 37oC for 2 hrs.
8) Destroy template by adding 2 ul of DNase I and incubating at 37oC for 15 min.
9) Hydrolyze probe by adding 20 ul of diw and 40 ul of carbonate buffer and incubating at 65oC for 40 min.
10) Neutralize with 80 ul of stop solution.
11) Add 16 ul of 4 M LiCl; 10 ul of glycogen, and 400 ul of EtOH. Mix and freeze at -80oC for at least 10 min.
12) Spin hard for 15 min. Rinse pellets with 70% EtOH.
13) Resuspend probes in 100 ul of in situ hyb buffer. Quantitate by dot blot using digoxigenin labelled RNA standards from BMB. Store probes at -20oC or in small aliquots at -80oC.
We typically use these probes at 0.5-1 ul/100 ul hybridization.