Protocol - Preparation of Digoxigenin labeled riboprobes
Preparation of riboprobes useful for in situ hybridization
Category:
Molecular Biology
Creation date: Jan 09, 2006
Last revision: Jan 20, 2015
Contact
Name: F. Anna Digilio
Address: Institute of Biosciences and BioResources - CNR, Via P. Castellino 111, 80131 Naples, Italy
Phone: +39 0816132430
Email: anna.digilio@ibbr.cnr.it
Steps
| Description | Temperature | Time | Note |
|---|---|---|---|
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Linearize plasmid with a restriction enzyme that cuts at the 5’ end of the insert
|
37°
|
1 hour
|
|
|
Analyze produc by gel electrophoresis to be sure that it is completily linearized.
|
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Add 1 ug of linear template to a sterile eppendorf on ice
|
|||
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Set up a 20 ul reaction on ice as reported below
|
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Mix briefly
|
|||
|
Incubate the reaction
|
37°
|
2 hours
|
|
|
Add RNase-free, Dnase I (2u) and incubate
|
37°
|
37°
|
optional
|
|
Stop the reaction by adding 1 ul of 0,5 M EDTA
|
(optional)
|
||
|
You can use one of two alternative options to hydrolize and precipitate the riboprobe. Consider that it is only necessary to hydrolyze probe if probe is greater than 500 bp – 1 kb. The hydrolysis of the probe allows for easier penetration of the probe into the tissue.
|
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Option A
|
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Hydrolize the probe by adding sterile water to 100 ul and 100 ul of 2x carbonate buffer and incubate
|
60°
|
10-120 min
|
probe hydrolysis times vary according to transcript length and can be calculated by allowing ca 15 min per 500 nucleotides.
|
|
Neutralize by adding 200 ul of 2x Hydrolysis-neutralization buffer
|
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Precipitate the RNA by adding 3 vol of cold 100% ethanol
|
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|
Mix well and incubate
|
-70°
|
30 min
|
|
|
Centrifuge 13000 g
|
4°
|
15 min
|
|
|
Wash the pellet in 100 ul of cold 70% ethanol.
|
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|
Centrifuge 13000 g
|
4°
|
15 min
|
|
|
Remove ethanol and air-dry the pellet
|
RT
|
5-10 min
|
|
|
Resuspend in 100-200 ul Rnase-free water
|
|||
|
Store the riboprobe
|
-70°
|
For hybridization use 0.5-1 ul per 100 ul of hybridiazation buffer
|
|
|
Option B
|
|||
|
Add 2 ul of LiCl 5 M and 80 ml of chilled absolute ethanol
|
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|
Mix well and incubate
|
-70°
|
60 min
|
|
|
Centrifuge 13000 g
|
4°
|
15 min
|
|
|
Wash the pellet in 100 ul of cold 70% ethanol.
|
|||
|
Centrifuge 13000 g
|
4°
|
15 min
|
|
|
Remove ethanol and air-dry the pellet
|
4°
|
10-15 min
|
|
|
Resuspend in 100 ul of Hybridization Buffer
|
4°
|
Although the probe will dissolve quickly in dep H20, it dissolves considerably more slowly in Hyb Buffer. Therefore, check to insure that it is dissolved.
|
|
|
Incubate
|
80°
|
10-30 min
|
probe hydrolysis times vary according to transcript length and can be calculated by allowing ca 15 min per 500 nucleotides.
|
Other informations
In our works, we used this protocol with the option B to obtain our riboprobes.
Quality validation:
Yes
Validation info
The protocol has been used to obtain data published in peer-reviewed journals.
Funded by: Grants from the Regione Campania (Law n. 5)
