Klymkowsky Lab On-line Methods
In situ hybridization - probe synthesis

Table of Methods updated - 10 May 2002

  1. Linearize 10 µg of plasmid using the appropriate restriction enzyme - do this for both antisense and sense probes. 
  2. Stop the reaction by heating the DNA at 65°C for 10 minutes. 
  3. Purify plasmid DNA by adding equal volume of 50% chloroform/50% phenol 
       (spin down for 5 minutes at room temperature and remove top layer). 
  4. Ethanol precipitate (for a 20 µl add 2.2 µl of NaOAcetate and 20 µl EtOH, 
        chill at -20°C for at least 30 minutes, 
        spin for 15 minutes (at maximum speed) and  re-suspend pellet in 20
    µl DEPC'ed H2O.
  5. Using Megascript Kit to make RNA.
  6. Thaw 10X transcription buffer, ribonucleotide solutions and RNAase-free water, 
        vortex, and microfuge to remove solution from the caps of the tubes  -- WEAR GLOVES! 
  7. Add the following amounts of the indicated reagents in the order below 
        to a 1.5
    µl RNase free microfuge tube at room temperature 

2 µl   10X transcription buffer 
µl   Ribonucleotide mix (10mM ATP, CTP, GTP, UTP & digoxigenin-UTP {2:1}) 
µl   Linearized plasmid DNA (1µg) (e.g. 2 µl of a 0.5 µg/ml template). 
µl    Enzyme mix 
µl    RNAase free water (final volume of reaction will be 20 µl

Mix and spin down 

8. Incubate in a cabinet 37°C incubator for between 2 to 6 hours 
   (if you do not have time to finish, freeze the reaction and resume the next day 
   - do not let the reaction go overnight!). 
   At this point you can run (on a 1% MOPS RNA gel)  1
µl of the reaction to see if you have probe. 

9. Add 1 µl  RNAase-free, DNAase I (2U/µl), mix,  spin and incubate at 37°C for 15 minutes.

10. Add 1/3 volume of 10 M LiCl in DEPC'ed H2O  mix thoroughly
- chill reaction for at least 30 minutes at -20°C

9. Centrifuge at 4°C for 25 minutes at maximum speed to pellet the RNA, 
    carefully remove the supernatant and wash the pellet with 70% ethanol. 
    Centrifuge at 4°C for 10 minutes at max speed to remove nucleotides. 

10. Remove as much supernatant as possible being 
    careful not to dry the pellet too much. 
     Resuspend in 20-100
µl DEPC-water and store at -80°C. 

Table of Methods