Dig-Riboprobe

Digoxigenin Riboprobe

Back to Vogt Lab Notes
Biology
Zebrafish

This follows exactly the recommended protocol from Boeheringer-Mannheim's "DIG RNA Labeling Kit" Cat. No. 1175025. Boeheringer's products are excellent, however, we found ourselves replacing individual components until all we were purchasing from Boeheringer was the dig-UTP. Protocol covers 2 ways we prepare DNA template (plasmid prep and PCR) and the general synthesis protocol.

Example (clone in pBluescript):


   M13    T7   PCR or    
   -40   site   cut v      insert
   ------------------xxxxxxxxxxxxxxxxxxxxxx-----------------
                                      cut ^    T3      M13        
                                      or PCR   site    rev

Stratagy:
DNA must be truncated on distal side of insert, depending on which enzyme you use. Otherwise you will make circular RNA. Accomplish this by either (1) preparing plasmid DNA and linearizing the DNA with an appropriate restriction enzyme (note warning below) or (2) generating DNA template by PCR using one vector primer outside the RNA pol site and the other preferably within the insert.

Problem with 3' overhangs: T3 and T7 RNA polymerases will wrap around a 3' overhang. This is not a significant problem with PCR products: Taq has terminal transferase activity which adds an A to the 3' ends, creating a single base 3' overhang. I used to polish (blunt end) my products, but have stopped doing this.

Amount of template: the reaction uses 1 ug of template. This does not include DNA that will NOT be incorporated into probe. Calculate template as insert. That is, a 500 bp insert in pBluescript requires 7 ug of plasmid/insert to contribute 1 ug of insert.

PCR Template (based on example above).

2. Clean products with phenol/chloroform, chloroform, Na-acetate precipitation. Dissolve final pellet in TE. OK to store frozen (-20 or -80).

3. Determine concentration.

4. Use 1 ug of product in recommended reaction mix.

Plasmid Template

2. Linearize plasmid with appropriate enzyme on the distal side of the insert from the desired enzyme promoter.

3. Clean by phenol/chloroform as above, dissolve final pellet in TE.

4. Use enough plasmid to provide 1 ug of insert in recommended reaction mix.

Reaction
We have been using T7 and T3 RNA polymerases from Stratagene (5/96); 50 U/ul. The same transcription buffer works for both enzymes. A 1X reaction yields about 10 ug RNA, a 2X reaction yields about 20 ug RNA; reagents are doubled EXCEPT the template.

                         ul    ul
                         1X    2X

                H2O       2    14
          5X buffer       4     8
digUTP labeling mix       2     4
   RNasin (Promega)       1     2
                DNA      10    10
             Enzyme       1     2

       total volume      20ul  40ul

Incubate: 37C, 2 hrs

Precipitate:
2.5 ul 8M LiCL (per 20 ul reaction)
55 ul EtOH (per 20 ul reaction)

Rinse with 300 ul 70% Ethanol (in DEPC H2O)
Dissolve pellet in 50 ul DEPC H2O.
Store -70C.
Examine 1-2 ul on1.5% Formaldehyde gel.

Digesting Probe.

Riboprobe can be alkaline degraded to around 150 bases by the following proceedure.

Stratagy:
An equal volume of RNA and digestion solution are mixed and incubated at 60oC for an appropriate time (30-45 min). Digested product is precipitated, resuspended in DEPC H2O and stored as usable probe at -70C.

Digestion solution:

Digestion time is determined by the following formula:
Digestion Time (min) = [Li-Lf]/[0.11*Li*Lf]

Reaction: 1. Equal volumes RNA and digestion solution (i.e. 50-100 ul each).
2. 60C for appropriate time (ca. 45 min).
3. Stop and precipitate:

4. 70 % EtOH wash.
5. 50-100 ul DEPC H2O (I use 50ul for a 1X rxn, 100 ul for 2X).
6. Store -70C.
7. Check aliquot (2-4 ul) on 1.5% formaldehyde gel against appropriate markers.