寡核苷酸的相关操作

 

 

 

In this section, you will find techniques related to oligonucleotides, such as oligo purification by acrylamide gel, annealing two oligos to make double stranded DNA and visualization by UV shadowing and more... 

Basics

・         Resuspending Lyophilized Oligos (IDT)

・         Preparation of oligonucleotide primers (GSC)
Prepare working primer solution from commercial synthesized oligos

・         Storage and Handling (Genosys)
Proper storage of your oligonucleotide will maximize its shelf-life, allowing you to get the most use from your oligo. Following these simple dilution and weight/volume unit conversion guidelines will enable you to work easily with your Genosys oligos.

Annealing of Oligos

・         Anneal Two Oligos to Make Double-Stranded DNA (IDT)
It is sometimes necessary to make double stranded DNA from single stranded oligos. While the annealing procedure is very simple, attention to a few details can greatly reduce the presence of undesired single stranded material.

・         Anneal Oligonucleotides to Make Double-Stranded DNA (Genosys)

・         Protocol for Adapter Production (LTI)
Anneal two oligos to make adaptor

Oligo Visualization

・         Oligonucleotide Visualization  
Visualize oligos by UV shadowing or methylene blue staining

・         UV Shadowing (Ambion)
UV shadowing is a technique for visualizing nucleic acids, e.g. separated in a gel, using short-wave UV light (254 nm) and a fluor-coated TLC plate. This method is for identifying nonisotopic probes or unlabeled RNA (e.g. for microinjection) during gel purification since the other alternative of staining with EtBr or acridine orange requires subsequent extraction of the dye. However, this method can be used to visualize any nucleic acid present in sufficient amount (> 0.3 µg).

Oligo Purification

・         Oligo Purification on Acrylamide Gels (PMCI Research)

・         Isolation of DNA from Acrylamide Gels (Schimenti Lab)

・         PAGE Purification of Oligonucleotides (LTI)
Protocol for PAGE Purification of Oligonucleotides Protocol for PAGE Purification of Oligonucleotides Oligonucleotides contaminated by significant amounts of aborted synthesis products (e.g., n-1,...

・         Oligonucleotide purification (Frank Bottone Jr.)
Buanol precipitation method

・         Butanol Precipitation of Deprotected Oligonucleotides (PMCI Research)
Crude oligonucleotides can be precipitated after deprotection directly from the ammonia solution without the need for a separate step to remove the ammonia. This procedure removes small molecular weight impurities such as the ammonia itself and the by products of deprotection (benzamide, isobutyramide). 

Oligo Labeling

・         Labeling of Oligonucleotides (PMCI Research)
Label oligo with polynucleotide kinase.  

・         Alkaline phosphatase labeling of oligos (Promega)
Provides useful guide for preparing phosphatase conjugated oligos 

・         5'-End Oligo Fluorescence Labeling (Amersham Pharmacia)
Labeling of  oligonucleotides with a single fluorescein residue at the 5' end

・         5' end labeling of oligonucleotides with gamma-³² P-ATP (Petra Klaff)
Labeling and purification method in great detail

Oligo Quantitation, Conversions and Calculations

• Common Conversions of Oligonucleotides (Fermentas)
    
• Melting Temperature of Duplex DNA & Oligonucleotides (Fermentas)
    

・         Micrograms of Double-Stranded DNA to Picomoles (Promega)

・         Picomoles of Double-Stranded DNA to Micrograms and Nanograms (Promega)

・         Micrograms per Milliliter of Oligonucleotides (Single-Stranded DNA) to picomoles per microliter (Promega)

・         Picomoles per Microliter of Oligonucleotides (Single-Stranded DNA) to micrograms per milliliter (Promega)

・         Micrograms of Linear DNA to Picomoles of Ends (Promega)

・         Molar Ratio of Insert to Vector for Ligations (Promega)

・         Conversion Factors for Metric Prefixes (Promega)

・         Oligonucleotide Melting Temperature Calculations (Promega)

・         Quantitation of Oligonucleotides (IDT)
Quantitate your oligo by UV Spectrophotometry or polyacrylamide gel

・         Quantitation of Oligonucleotides (Genosys)
UV Quantitation

・         Calculation of  Melting Temperature (Genosys)

Oligonucleotide Calculator
Determine Tm, MW, etc. of your oligonucleotide (requires &#106a;ascript enabled