Post Electrophoretic Analysis Articles
Gel Preparation for Native PAGE of DNA
Native PAGE gels are prepared by mixing an acrylamide/bisacrylamide monomer concentrate (AccuGel 19:1 or 29:1), buffer concentrate, and water to achieve the desired gel concentration. TEMED and ammonium persulfate are added to initiate polymerization and the solution is poured into a cassette. The comb is then inserted.
The cassette is formed by two glass plates separated by spacers, typically 0.5-1mm in thickness, and sealed at the bottom (See figure below or follow this link for a more detailed discussion of the electrophoresis apparatus). The polymerization process takes 1-2 hours to complete.
The table below gives solution amounts to prepare gels of various percentages and buffers.
Instructions:
- Prepare a solution of monomer, buffer, and water as per the table.
- De-gas if desired.
- Add APS and TEMED.
- Pour gel.
Gel % | TBE Gels | TAE Gels | TTE Gels |
---|---|---|---|
6 | AccuGel: 15 mL 10X TBE: 10 mL Deionized H2O: 75 mL |
AccuGel: 15 mL 50X TAE: 2 mL Deionized H2O: 83 mL |
AccuGel: 15 mL 20X TTE: 5 mL Deionized H2O: 80 mL |
8 | AccuGel: 20 mL 10X TBE: 10 mL Deionized H2O: 70 mL |
AccuGel: 20 mL 50X TAE: 2 mL Deionized H2O: 87 mL |
AccuGel: 20 mL 20X TTE: 5 mL Deionized H2O: 75 mL |
10 | AccuGel: 25 mL 10X TBE: 10 mL Deionized H2O: 65 mL |
AccuGel: 25 mL 50X TAE: 2 mL Deionized H2O: 78 mL |
AccuGel: 25 mL 20X TTE: 5 mL Deionized H2O: 70 mL |
12 | AccuGel: 30 mL 10X TBE: 10 mL Deionized H2O: 60 mL |
AccuGel: 30 mL 50X TAE: 2 mL Deionized H2O: 73 mL |
AccuGel: 30 mL 20X TTE: 5 mL Deionized H2O: 65 mL |
14 | AccuGel: 35 mL 10X TBE: 10 mL Deionized H2O: 55 mL |
AccuGel: 35 mL 50X TAE: 2 mL Deionized H2O: 68 mL |
AccuGel: 35 mL 20X TTE: 5 mL Deionized H2O: 60 mL |
NEXT TOPIC: PCR Analysis: An Examination
- Using PAGE to Determine Nucleic Acid Molecular Weight
- SSCP Analysis
- Sanger Sequencing
- Sample Preparation for Native PAGE of DNA
- Sample Prep for Denaturing PAGE of DNA
- S1 Mapping
- Run Conditions in Denaturing PAGE
- RNA Mapping
- RNA Electrophoresis
- Ribonuclease Protection
- Restriction Digest Mapping
- Primer Extension
- Preparing Denaturing DNA & RNA Gels
- Preparation of Denaturing Agarose Gels
- Preparation of Agarose Gels
- Pouring Sequencing Gels
- PFGE and FIGE
- PCR Analysis: Yield and Kinetics
- PCR Analysis: An Examination
- Native PAGE of DNA
- Mobility Shift Assay
- Methylation & Uracil Interference Assays
- Maxam & Gilbert Sequencing
- Manual Sequencing
- In Gel Enzyme Reactions
- Heteroduplex Analysis
- Gel Preparation for Native PAGE of DNA
- Gel Electrophoresis of PCR Products
- DNase I Footprinting
- DNA/RNA Purification from PAGE Gels
- DNA/RNA Purification from Agarose Gels – Electroelution
- Differential Display
- Denaturing Polyacrylamide Gel Electrophoresis of DNA & RNA
- Conformational Analysis
- Automated Sequencers
- Analysis of DNA/Protein Interactions
- Agarose Gel Electrophoresis of DNA and RNA – Uses and Variations
- Agarose Gel Electrophoresis of DNA and RNA – An Introduction