Experiments on purification of oligonucleotides by preparative gel electrophoresis
Experiments on purification of oligonucleotides by preparative gel electrophoresis
This experiment describes how to purify oligonucleotides by preparative gel electrophoresis. This experiment is from Protein Purification and Identification Laboratory Guide by Houzhu Zhu.
Operation method
Experiments on purification of oligonucleotides by preparative gel electrophoresis
Materials and Instruments
Acrylamide Bisacrylamide Urea Ammonium Persulfate N N N' N'-Tetramethylethylenediamine Synthetic Complementary Oligonucleotides Sertiary Butyl Alcohol (2-Butanol) Diethyl Ether NaOAc MgCl2 Ethanol TBE Formamide Spiking Buffer TE Move Materials and equipment For more product details, please visit Aladdin Scientific website.
SpeedVac Rotary Concentrator
Acrylamide
Bisacrylamide
Urea
Ammonium persulfate (10%;w/v)
N,N,N',N'-Tetramethylethylenediamine (TEMED)
Synthesized complementary oligonucleotide (containing the recognition sequence of the DNA binding protein specific to the sequence to be isolated)
sec-butanol (2-butanol)
diethyl ether
NaOAc(3mol/L)
MgCl2 (1mol/L)
Ethanol (100% and 75%; v/v)
SpeedVac Rotary Concentrator (Savant Instruments, Inc.)
Reagents
TBE (10x)
Formamide Spiking Buffer
TE
(For the recipe, see "Preparation of Reagents", P.131~138.)
Operating Procedures
Preparation of polyacrylamide gel
1) Prepare a 20-cm x 40-cm x 1.5-mm gel with four 3-cm wide sample wells. For oligonucleotides in the length range of about 10-45 bases, a 16% polyacrylamide/urea gel is preferred. For longer oligonucleotides, 8% or 6% polyacrylamide/urea gels may be used.
2) To prepare a 16% polyacrylamide/urea gel, mix as follows:
Acrylamide (38%; w/v)/bisacrylamide (2%; w/v) 50 ml
TBE (l0x) 12.5 ml
Urea 62.5 g
H20 17 ml
Total volume 125 ml
3) Filter, degas briefly, then add 750ul 10% (w/v) ammonium persulfate and 50ul TEMED.
4) Allow the gel to polymerize for ≥30 min, then pre-electrophorese the gel for at least 1h at 30W.
Note: Pre-electrophoresis of the gel helps to remove excess persulfate ions, which can degrade DNA.
5)-The amount of DNA that can be added to a gel should not exceed 1umol (~1-2 mg) of synthetic oligonucleotide (all 4 3-cm wells were used, each containing 0.25umol of sample). This amount of DNA is approximately the maximum amount that can be added to the gel without overloading it.
Note: The mobility of DMA in 16% gels is as follows: Bromophenol Blue will co-migrate with oligonucleotides up to 10 bases in length, Xylene Nitrile Blue will co-migrate with oligonucleotides up to 30 bases in length, and it is recommended that only Bromophenol Blue be added to the Formamide Sampling Buffer for oligonucleotides that are 25-35 bases in length.
Sample Preparation
1) Dissolve the oligonucleotides in appropriate amount of formamide spiking buffer, just enough to add 50ul of sample into a 3-cm well.
2) Heat the sample at 65℃ for 15 min to eliminate the secondary structure in the DNA.
3) Add sample. The gel was electrophoresed at 30W. It takes about 4 h for the bromophenol blue to travel down to 3/4 of the gel, which is sufficient to purify oligonucleotides 15-30 bases long.
Recovery of DNA
1) Ultraviolet (UV) imaging to identify the main band (usually the largest oligonucleotide, but sometimes the next largest).
Note: UV contrast is performed as follows. Place the polyacrylamide gel between two sheets of plastic wrap (e.g., Sarnn wrap) and place the gel on a thin plate containing fluorescent material (e.g., a thin-layer chromatography plate containing fluorochromes or a radiographic autoradiography screen). A handheld UV lamp is used to shine long wavelength UV light on the gel (for a short time!) . Fluorescence will be observed everywhere on the plate of fluorescent material except where the DNA band is located (because DNA absorbs UV light). As a result, a shadow will appear under the DNA band. Make a quick outline around the DNA band with a pen and then cut the corresponding part of the gel containing the DNA under normal light.
2) Carefully cut off the DNA strip with a razor blade, trying to avoid shredding the gel (do not break the strip into small pieces).
3) Soak the gel block in 5 ml of TE buffer (in a 15-ml polypropylene tube) and shake at 37°C overnight.
4) Filter the supernatant through silanized glass wool in a pasteurized pipette.
Note: It is important that the glass wool in the pasteurized pipette is pre-soaked in approximately 5 ml of water.
5) Concentrate the DNA by repeated extraction with sec-butanol.
6) Extract the DNA with diethyl ether - once - and remove the residual ether under vacuum with a SpeedVac rotary concentrator.
7) Bring the volume of liquid to 180ul with TE buffer.
8) Add 20ul of 3mol/LNaOAc and 2ul of 1mol/LMgCl2, shake and mix well.
9) Add 600ul 100% ethanol and mix upside down. Chill in dry ice/ethanol (-78°C) for 10 min. leave the mixture at room temperature for 5 min. centrifuge at high speed for 15 min at room temperature to precipitate the DNA.
10) Add 180ul of TE buffer and shake to dissolve the precipitate.
11) Add 20ul of 3mol/LNaOAc and 2ul of 1mol/LMgCl2, shake and mix well.
12) Add 600ul of 100% ethanol, invert and mix. Cool the mixture in dry ice/ethanol (-78°C) for 10 min. leave the mixture at room temperature for 5 min. centrifuge the mixture in a microcentrifuge at high speed for 15 min at room temperature to precipitate the DNA.
13) Add 800ul of 75% ethanol to precipitate DNA, shake well. Centrifuge for 5 min at room temperature.
14) Carefully dry the precipitate with a SpeedVac rotary concentrator (caution: sometimes static electricity can cause the precipitate to fall out of the centrifuge tube).
15) Dissolve DNA in TE buffer and store at -20 °C.
16) Measure A260nm and A280nm.
Note: For sequence-specific DNA affinity media, assume 1A260nm units = 40ug/ml DNA.