Protocol - RNA riboprobe synthesis
T7 RNA polymerase transcription
Category:
Molecular Biology
Last revision: May 22, 2015
Author(s):
Laura Pisapia, Pasquale Barba, Giovanna Del Pozzo
Contact
Name: Giovnna Del Pozzo
Address: IGB- CNR
Phone: 0816132309
Email: giovanna.delpozzo@igb.cnr.it
Steps
| Description | Temperature | Time | Note |
|---|---|---|---|
|
Prepare DNA template by PCR
|
3-4 hrs
|
||
|
Use 0,5 ug of template to perform T7 RNA in vitro transcription by MaxiScript T7 kit ( AMBION) in presence of 40 uCi 32P UTP
|
1,5 hrs
|
||
|
Purify the RNA labelled riboprobe by micro Bio Spin Column P30 Tris RNase free ( BIORAD)
|
15 min
|
||
|
Measure 32UTP incorporation by beta-counter
|
30 min
|
Quality validation:
Yes
Validation info
The protocol has been used to produce data published in peer-reviewed journals.
