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Digoxigenin-labeled riboprobe synthesis from plasmid template (Version 1.0) | ATLAS-D2K Center

PLEASE NOTE: ATLAS-D2K closed July 31, 2025 and this website is for reference purposes only.

Digoxigenin-labeled riboprobe synthesis from plasmid template (Version 1.0)

Version

1.0

Notice

This page is the corresponding protocol tomestone page generated as part of the ATLAS-D2K shutdown in July 2025. Many links on this page may be broken.

Authors

Martin Cohn

Keywords

[‘plasmid’]

Subjects

[‘Developmental biology’, ‘Gene expression analysis’, ‘Molecular biology’]

Release Date

2017-08-07

Abstract

Digoxigenin-labeled riboprobe synthesis from plasmid template protocol by the Cohn Group

Procedure

See PDF.

Plasmid Preparation

All plasmids are I.M.A.G.E clones which can be obtained from Open Biosystems http://www.lifesciences.sourcebioscience.com.

Sterile conditions are maintained at all times when working with bacteria and the glycerol stocks.

Day 1: Plating out from a glycerol stock

  1. Take the culturing plates out of the fridge and let them warm up at room temperature for 15-30 minutes.

  2. Ensure that the plates contain the correct antibiotic to select for the bacteria containing the recombinant plasmid. Kanamycin (Kan) and chloramphenicol (Cam) can be added directly to non-ampicillin plates. Add 20µl of Kan (50mg/ml) or Cam (25mg/ml) on to the plate and spread.

  3. Spread 4µl of 25mg/ml Xgal for the blue/white colony screen.

  4. Streak plate with the glycerol stock using a sterile pipette tip.

  5. Incubate upside down overnight at 37°C.

Day 2: Mini Culture

  1. Set up a small culture in the late afternoon.

  2. Select a white colony using a sterile pipette tip and transfer in to 2ml Terrific Broth (TB) with the appropriate concentration of antibiotic added.

  3. Incubate overnight at 37°C in an orbital shaker set at 225rpm.

Day 3: Midi Culture

  1. Store the 2ml culture at 4°C until later in the afternoon.

  2. Add 2mls of this culture to 50ml TB containing the appropriate concentration of antibiotic.

  3. Incubate overnight at 37°C in an orbital shaker set at 225rpm.

Day 4: Midi plasmid preparation

  1. Pour the bacterial culture in to a 50ml centrifuge tube.

  2. Pellet the bacteria by centrifugation. Centrifuge: Avanti J26-XP, Beckman Coulter Centrifuge at ≥20,000 x g.

  3. Drain the supernatant. The pellet can be frozen and stored at -20°C.

  4. Extract the plasmid DNA using a QIAGEN Plasmid Midi Kit according to the manufacturer’s instructions.

  5. Re-suspend the plasmid DNA in 100µl of TE buffer (pH 8.0).

  6. Determine the yield, DNA concentration & purity of the plasmid DNA using spectrophotometry at 260 nm. For further qualitative and quantitative analysis run a small aliquot of plasmid DNA on a 1%** agarose gel.

Day 5: Plasmid template linearization

  1. Set up the following restriction digest to linearize the plasmid template.

    Plasmid				20µg		

Restriction enzyme		2µl

Restriction buffer		10µl

Make up to 100 µl with H2O

  2. Incubate overnight at the appropriate temperature for the restriction enzyme digest.

Day 6: Restriction digest clean up with phenol:chloroform

  1. Add 1 volume of phenol:chloroform (50:50 mix) to the digest.

  2. Vortex and centrifuge at 13K rpm for 1min in a bench centrifuge.

  3. Transfer the aqueous layer to a clean tube (discard organic layer).

  4. Add 1 volume chloroform, vortex and centrifuge at 13K rpm for 1min.

  5. Transfer the aqueous layer to a clean tube.

Precipitate DNA

  1. Vortex.

  2. Add 2.5 volumes ethanol, mix by inversion and place on dry ice for 10-20 minutes.

  3. Centrifuge in the cold room at 13K rpm for 15 minutes.

  4. Aspirate supernatant taking care not to lose the pellet

  5. Wash pellet by adding 70% ethanol and vortex.

  6. Centrifuge at 13K rpm for 10 minutes.

  7. Carefully aspirate the ethanol.

  8. Pulse spin to bring down remaining ethanol and aspirate again to remove the alcohol.

  9. Leave the tube open on the bench for 15 minutes to allow the ethanol to evaporate from the pellet.

  10. Resuspend pellet in 20µl DEPC-treated H20.

  11. To check the quality and quantity of the linearized DNA load 1µl of DNA with 4 µl loading buffer and run on a 1% agarose gel. If band is a single clean band label the probe on the same day.

In vitro transcription of digoxigenin-labeled riboprobes

Ensure RNase-free conditions are maintained and that the reagents are kept on ice.

  1. Mix together the following:

    DEPC H2O			Xµl*
	
5X T3/T7/Sp6 buffer		4µl
	
DTT				2µl (Promega #P1171)
	
DigUTP Nucleotide mix		2µl (Roche #11277073910) 
	
DNA (1µg)			Xµl*
	
Placental Rnase inhibitor	0.5µl (Promega #N2511)
	
T3, T7 or Sp6 enzyme		2µl (Promega, T3 #P4024, T7 #P4074 or Sp6 #P4084)

* Adjust DNA and H2O to give a 20µl total volume.

  2. Incubate overnight at 37oC.

  3. Add 80µl RNase free water

  4. To extract the RNA use mini Quick Spin RNA Columns (Roche #11814427001).

  5. Determine the yield, concentration & purity of the RNA using spectrophotometry at 260 & 280nm.

Media & Solutions

Terrific Broth (TB) with salts, no amp (1l)

  • 12 g tryptone (Fisher Scientific #BP1421500)

  • 24 g yeast extract (Fisher Scientific #BP1422500)

  • 4 ml glycerol (Fisher Scientific #BP229-4)

  • Dissolve to a final volume of 900 ml with ddH2O

  • Autoclave at 121°C for 45 minutes.

  • Cool to touch on stir plate.

  • Add 100 ml 10X TB salts.

  • Store 50 ml aliquots at -20°C.

10X TB salts (250ml)

  • 5.78 g KH2PO4

  • 31.35 g K2HPO4

  • Dissolve to a final volume of 250 ml with ddH2O

  • Autoclave at 121°C for 45 minutes.

Antibiotic Stock Solutions

  • Ampicillin 60mg/ml

  • Kanamycin 50 mg/ml

  • Chloramphenicol 25 mg/ml

  • Add antibiotic to the media at a concentration of 1µl/ml.

Luria Broth (LB)+ Ampicillin plates

  • Add 1l ddH2O to 2l flask with stir bar.

  • Add 40 g LB Agar (Acros #61189-5000)

  • Stir until dissolved.

  • Autoclave at 121°C for 45 minutes.

  • Cool to touch on stir plate.

  • Add ampicillin to a final concentration of 100 mg/l.

  • Immediately pour plates and allow to cool (keep a flame going).

  • Once cool, cover and mark sizes with black (LB) and red (amp) lines.

  • Store in plastic sleeve with lids facing down at 4°C.

TE Buffer pH 8.0

  • 1mM Tris pH 8.0
  • 1mM EDTA

For 500ml:

  • 1M Tris pH 8.0 (5ml)
  • 0.5M EDTA pH 8.0 (1ml)
  • dH2O 496ml

Loading Buffer

Gel loading dye, Blue 6X (New England BioLabs #B7021S)

Consortium

GenitoUrinary Development Molecular Anatomy Project (GUDMAP) Consortium