Protocol - Preparation of radioactive probes for nucleic acids blots
It generates highly specific and hot probes.
Category:
Molecular Biology
Creation date: Jun 18, 2014
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: +39 081 6132430
Email: anna.digilio@ibbr.cnr.it
Steps
| Description | Temperature | Time | Note |
|---|---|---|---|
|
Prepare DNA template (300-600 bp) by PCR/restriction + gel purification
|
|||
|
Prepare a normal PCR reaction with the final concentrations ot the radionuclide as reported below
|
Parameters: Reaction volume: 25 ul; Taq: e.g. Roche, 1U for 25 μl rxn; DNA: 2 ng/ul rxn; Primer: 1 μM each
|
||
|
Start a PCR cycle as reported
|
|||
|
No need for denaturing
|
|||
|
You can also use just one primer in order to obtain a probe complementary to the mRNA
|
the primer should be internal to that used for preparing the template through PCR.
|
Other informations
Radionuclide final concentrations:
|
Typea[cold:hot] |
dAGTTP* uM each] |
Cold dCTP [uM] |
Hot dCTP [uM] |
ul of standard (3.33 uM) hot dCTP in 25ul |
|
Low [2:1] |
50 |
0.7 |
0.33 |
2.5 |
|
High [1:1] |
50 |
0.7 |
0.67 |
5 |
a 1:1 for sensitive applications.
* also 100 uM worked.
|
Standard radionuclide: |
32P, alpha position, dCTP |
10 uCi/ul |
3000 Ci/mmol (= 3.33 uM) |
colored buffer |
- Cycling:
- 1x 94°C 1’
- 40-60x (usually 50x)
- 94°C 20’’
- An.T 30’’
- 72°C 1’30’’-2’ (6’40’’/kb or 0.4’’/nt with 100 uM dAGT-tp and Taq)
Quality validation:
Yes
Validation info
The protocol has been used to obtain data published in a peer-review journal.
Funded by: Grant from the Regione Campania (L.R. 31.12.1994 n.5)
