I need to make a DNA strand consisting of two 35 nt sticky ends and 147 bp of double-stranded nucleic acid.I achieved this with primers that added spacer. However, there are still a large number of non-specific products after purification by PEG. How can I improve my protocol?
you could cut the good band out of the gel and use a gel purification kit (Qiagen or similar)
or if this is not available excise the band into a small tube,freeze thaw it 3 times then spin down hard and the supernatant should be your required product
Purifying DNA depends on your source material and the level of purity required. Here are some general methods:
1. DNA Extraction from Cells or Tissues
If you’re extracting DNA from cells, plants, or animal tissues, follow these basic steps:
- Cell Lysis: Break open the cells using a detergent (e.g., SDS) or enzymatic digestion (e.g., proteinase K).
- Protein Removal: Use phenol-chloroform extraction or proteinase treatment.
- DNA Precipitation: Add cold ethanol or isopropanol to precipitate DNA.
- DNA Wash and Resuspension: Wash with ethanol and dissolve in a buffer (e.g., TE buffer or nuclease-free water).
2. Commercial DNA Extraction Kits
These provide a standardized way to purify DNA with silica membranes or magnetic beads. Some common kits include:
- Qiagen DNeasy Kits
- Zymo Research DNA Kits
- Promega Wizard Genomic DNA Purification Kit
3. Column-Based Purification (Silica or Anion-Exchange)
- DNA binds to silica in the presence of high salt.
- Wash contaminants away.
- Elute with low-salt buffer or water.
4. Magnetic Bead-Based Purification
- DNA binds to coated magnetic beads.
- Wash away impurities.
- Elute in a clean buffer.
5. Gel Extraction for Pure DNA Fragments
If you're isolating specific DNA fragments, gel electrophoresis followed by gel extraction kits (or freeze-thaw methods) can help.
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