Overview
RTPCR Primers
RTPCR Protocol
ISH Probes
ISH Protocol

Oligo Design

Our suggestions for making oligoprobes and primers using the Oligo 5.0 software.

Oligoprobe Design
Primer Design
Oligo Step by Step
 
 

Oligoprobe Design

Placing: consider where a corresponding primer pair would be placed; published suggestions; splice sites; isoforms.
Molecular characteristics: length, melting temperature and GC-content should be kept similar for all probes designed so that the same protocol can be used in all experiments. Melting temperature should be set so that it is at least 15 above washing temperature(55°C). Hairpins should be minimized. Especially those with negative values (hairpin then forms spontaneously). Stable dimer formations should be avoided. All hairpins or dimers hiding the 3'-end of the probe will interfere with radionucleotide labeling.


Length:  GC-content Tm (melting temperature GC-method)
45-55 (48 recommended) 50-60% 80-95°C (95 at most)

Finishing
The following information should always be printed:
lower primer composition
lower primer duplexes
lower primer hairpin stems
(unless your sequence is inverted when the upper/current oligo info should be printed)


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Primer Design

Placing: should cover site for possible oligoprobe. Consider published suggestions; splice sites; isoforms. Remember that the inclusion of mRNA splice sites can result in the production of more than one product. Amplicons (the product) spanning introns allow for the possibility to detect DNA contamination in extracted mRNA template.Molecular characteristics: melting temperature of upper and lower primers should differ no more than 1°C. Hairpins at the 3'-end should be avoided since they can be sites for initiation of priming, thus causing primers to be eliminated from the PCR. Any dimer at the 3' end with free energy below -2 kcal/mol will inhibit the PCR reaction. Dimers and  hairpins at the 5' end are generally nonsignificant.


Primer Length:  Product length, bp GC-content Product Tm Annealing T
20-24 (shorter for real time-PCR) 350-700 (shorter for real time-PCR) 50-55% <90°C <60°C

Finishing
The following information should always be printed:
primer compositions
primer duplexes
primer hairpin stems
false priming sites


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Oligo Step by Step

Retrieve and open database
Using your references and GenBank accession numbers, locate the nucleotide sequence you want to make probes and primers for. At the bottom of the page choose text and then, in the file menu, save as a text file (name.txt). Open this text file separately and remove the ”//” at the end of the document. Save it. Alternatively, copy the text, excluding the ”//” at the end, and save it in eg notepad.
In Oligo, choose open database and open your text file.

Getting started
Once you have opened your database you can begin your search for primers and probes. Set your search ranges to match the cds (coding sequence) or to a region were you need to place your probe or primers and product.

Good to know about probe searching
Your database corresponds to the red ”upper primer” sequence and consequently your oligo should be identical to the coresponding ”lower primer” sequence. The search engine in oligo searches the database, ie the red sequence. Consequently the search is relevant only when it comes to Tm and GC% figures, since these are identical for the upper and lower sequences. However, the search engine will fail to detect lower sequence 3' hairpins and duplexes, that should exclude the oligo, since it is regarding these as positioned in the 5' end of the sequence it is searching in. Thus, it is always necessary to inspect these parameters visually, for all oligo suggestions. Alternatively the sequence can be inverted in the edit entire sequence menu. Doing this allows you to use the search engine efficiently. Remember to calculate your probe position correctly to match the original sequence or return it once you have found your probe.
 
 

Suggestions and Comments

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