Bruce´s Totally Anal RNA in situ Probe Synthesis Protocol

A. DNA Template Preparation

  1. Linearize 5-10 ug plasmid with enzyme (avoid enzymes that leave 3´ overhang)
  2. Following digestion, Add:
    5 ?l 10 mg/ml ProtK
    2.5 ?l 10% SDS.
  3. Incubate at 50° C for 1hr.
        Note: this step is to get rid of residual RNase from Qiagen preps.
  4. Add 50 ul DEPC-H2O, 10 ?l 3M NaOAc, pH 5.3.
  5. Phenol/chloroform extract
  6. EtOH ppt.
  7. Resuspend in DEPC-H2O at 500ng/ul

B. Probe Synthesis

  1. Assymble the following (in order):
    11 ?l DEPC-H2O
    2 ?l 10X Transcription Buffer
    2 ?l 10X DIG NTP mix
    2 ?l 500 ng/?l linearized DNA template (1?g total)
    1 ?l 100 mM DTT
    1 ?l RNasin
    1 ?l Appropriate RNA Polymerase

  2. Incubate at 37° C for 1hr
  3. After 1 hr, add additional 1?l of polymerase and incubate 1hr more
  4. Add 0.8 ?l 0.5 M EDTA
  5. Remove 1 ?l to analyze on gel
    6. (OPTIONAL: Purify RNA on Sephadex G-50 Spin Column)
  6. Precipitate RNA by adding:
    7. 1 ?l 50 mg/ml yeast tRNA
    2 ?l 5 M LiCl
    75 ?l EtOH

  7. Incubate at -20° C for 15 min.
  8. Spin 20 min at 14K in 4° C microfuge
  9. Rinse pellet with 70% EtOH
    10. (OPTIONAL: Hydrolyze long probes to smaller size)
  10. Resuspend in either 100 ?l DEPC-H2O, or in situ Prehyb buffer.
  11. Store at -80° C

C. Probe Purification using a Sephadex G-50 Spin Column (Optional)

Note: Probes purified over a G-50 spin column (Roche cat# 1 274 015) have much less background than un-purified probes. If background is not a problem, this step is not necessary.

Following Probe Synthesis but prior to precipitation (i.e. step 5):

  1. Add 30?l DEPC-H2O (Vt= 50 ?l)
  2. Purify over Sephadex G-50 spin column
  3. After recovery from column, bring volume up to 50 ?l
  4. Remove 2?l to analyze on gel
  5. Add 5?l 5M LiCl, 188?l EtOH and place at -20° C for 15 min.
  6. Spin 20 min. at 14K in 4° C microfuge.
  7. Rinse pellet with 70% EtOH
    (Optional: Hydrolyze long probes)
  8. Resuspend in 50?l DEPC-H2O or Pre-Hyb
  9. Store at -80° C

D. Probe Hydrolysis (Optional)

Note: For probes longer than 1.5-2.0 kb, signal:noise can be improved by hydrolyzing probe to a smaller size (~0.3 kb).
  1. Resuspend pellet from above in 40 ul DEPC-H2O
  2. Add:
    5ul 0.4M Na Bicarbonate (1.68 gm/50 ml)
    5ul 0.6M Na Carbonate (3.72 gm monohydrate/ 50 ml)

  3. Incubate at 60° C for t min.,
    where t min= (starting kb - desired kb)/0.11(starting kb)(desired kb)
    Note: A good "desired size" is 0.3 - 0.4 kb
  4. Add:
    200 ul DEPC-H2O
    17 ul 3M NaOAc
    1.3 ul Glacial HOAc
    550 ul EtOH (-20° C)

  5. Incubate at -20° C for 30min.
  6. Resuspend in 100 ul DEPC-H2O and store at -80° C
  7. Analyze 5ul on gel to access cleavage
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